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

Angiostrongylus dujardini infection in a coconut lorikeet (Trichoglossus haematodus) from a zoological garden in Switzerland.

Angiostrongylus spp. (Metastrongyloidea) can cause severe disease in several animal species and humans. This report describes an infection with Angiostrongylus dujardini in a captive Coconut lorikeet (Trichoglossus haematodus) from a zoo in Switzerland. The bird was reported being attacked by conspecifics, removed from the flock, and hospitalized. It showed lethargy, moderately reduced body condition, and lack of reaction to visual stimuli. Analgesic and antibiotic treatment were initiated but because of worsening of its general condition the bird was euthanized the following day. Necropsy revealed multifocal, subcutaneous hemorrhages, diffusely reddened lungs and a moderately dilated right heart with several intraluminal nematodes embedded in a coagulum. Four worms were collected and microscopically examined. They were identified as adult females, measuring 19-21 mm long x 0.4-0.5 mm wide, with general morphological and morphometric characteristics consistent with angiostrongylid nematodes. In lung sections, multifocal collection of thin-walled embryonated eggs in variable stages of development was observed along with fully developed nematode larvae within the lumina of alveoli and lung vessels. Associated granulomatous infiltrates indicated a severe, multifocal, chronic, granulomatous pneumonia. The diagnosis of A. dujardini infection was formulated by morphological examination of adult and larval stages, supported by molecular analysis (PCR-amplification and sequencing of the ITS2, 5.8S and 28S rDNA flanking regions). This is the first report of A. dujardini infection in an avian species, providing evidence that birds can serve as accidental hosts of this parasite in addition to mammals, and that the parasite can reach maturity and multiply in the avian cardiorespiratory system

Fatal gastritis and enterocolitis due to concurrent Helicobacter pylori and Campylobacter jejuni infection in a captive cheetah (Acinonyxjubatus).

A 3.5-year-old female cheetah (Acinonyx jubatus) died after a 10-day history of anorexia, regurgitation and diarrhoea despite symptomatic therapy. At gross post-mortem examination, the stomach was blood-filled with mucosal thickening and multifocal ulcerations. The intestinal mucosa was thickened and reddened, and the intestinal lumen was filled with dark red to black pasty content. Gastric histological lesions were compatible with gastritis due to Helicobacter infection, which was confirmed by polymerase chain reaction. Histology of the intestines revealed a severe necrotizing neutrophilic enterocolitis with abundant intralesional curved to spiral bacteria, corresponding to Campylobacter jejuni, which were subsequently isolated from both small and large intestinal contents. No other intestinal pathogens were detected despite thorough investigations. These findings suggest that C. jejuni may have played an aetiological role in the enterocolitis. Such an association has not been previously reported in non-domestic felids

Clostridium perfringens-Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus).

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets (P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined

Fatal gastritis and enterocolitis due to concurrent Helicobacter pylori and Campylobacter jejuni infection in a captive cheetah (Acinonyx jubatus)

A 3.5-year-old female cheetah (Acinonyx jubatus) died after a 10-day history of anorexia, regurgitation and diarrhoea despite symptomatic therapy. At gross post-mortem examination, the stomach was blood-filled with mucosal thickening and multifocal ulcerations. The intestinal mucosa was thickened and reddened, and the intestinal lumen was filled with dark red to black pasty content. Gastric histological lesions were compatible with gastritis due to Helicobacter infection, which was confirmed by polymerase chain reaction. Histology of the intestines revealed a severe necrotizing neutrophilic enterocolitis with abundant intralesional curved to spiral bacteria, corresponding to Campylobacter jejuni, which were subsequently isolated from both small and large intestinal contents. No other intestinal pathogens were detected despite thorough investigations. These findings suggest that C. jejuni may have played an aetiological role in the enterocolitis. Such an association has not been previously reported in non-domestic felids

CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts

Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin (VIM), which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis

Spongiform Encephalopathy in a Miniature Zebu

The first case of spongiform encephalopathy in a zebu (Bos indicus) was identified in a zoo in Switzerland. Although histopathologic and immunohistochemical analyses of the central nervous system indicated a diagnosis of bovine spongiform encephalopathy (BSE), molecular typing showed some features different from those of BSE in cattle (B. taurus)

Effects of Storage Time and Thawing Method on Selected Nutrients in Whole Fish for Zoo Animal Nutrition

Piscivores in human care receive whole fish that were frozen, stored and thawed before feeding. Nutrient losses have been documented, but exact changes during storage and with different thawing methods are unknown. Primarily, it was hypothesized that frozen fish lose different vitamins and trace minerals during a storage period of six months. Secondly, that different thawing methods have a significant influence on the degree of vitamin loss. Three fish species, herring (Clupeus harengus), mackerel (Scomber scombrus) and capelin (Mallotus villosus) were analyzed at four time points within a storage period of 6 months at −20 °C. At each time point, three thawing methods were applied: thawing in a refrigerator (R), thawing at room temperature (RT), and thawing under running water (RW). The following nutrients were analyzed: vitamin A, B1, D3 and E, iron (Fe), copper (Cu), zinc (Zn) and selenium (Se). The statistical method used was a linear mixed effect model. Cu was below detection limits in all analyzed samples, vitamin B1 in most analyzed herring (44/48 samples) and capelin (in 25/36 samples), respectively. In addition, the vitamin D3 concentration was also below detection limits in half of the capelin samples (18/36). No concentration changes of Fe (p = 0.616), Zn (p = 0.686) or Se (p = 0.148) were observed during a storage period of six months, in contrast to a significant decrease in vitamin A (p = 0.019), D3 (p = 0.034) and E (p = 0.003) concentrations. Thawing fish with different thawing methods did not result in concentration changes of Fe (p = 0.821), Zn (p = 0.549) or Se (p = 0.633), but in a significant concentration change of vitamin A (p = 0.002). It is essential to supplement vitamins B1 and E in diets containing whole fish to avoid deficiencies in piscivorous species, and care should be taken not to store fish longer than six months, due to the depletion of vitamins A, D3 and E

Comprehensive Serology Based on a Peptide ELISA to Assess the Prevalence of Closely Related Equine Herpesviruses in Zoo and Wild Animals

Equine herpesvirus type 1 (EHV-1) causes respiratory disorders and abortion in equids while EHV-1 regularly causes equine herpesvirus myeloencephalopathy (EHM), a stroke-like syndrome following endothelial cell infection in horses. Both EHV-1 and EHV-9 infections of non-definitive hosts often result in neuronal infection and high case fatality rates. Hence, EHV-1 and EHV-9 are somewhat unusual herpesviruses and lack strict host specificity, and the true extent of their host ranges have remained unclear. In order to determine the seroprevalence of EHV-1 and EHV-9, a sensitive and specific peptide-based ELISA was developed and applied to 428 sera from captive and wild animals representing 30 species in 12 families and five orders. Members of the Equidae, Rhinocerotidae and Bovidae were serologically positive for EHV-1 and EHV-9. The prevalence of EHV-1 in the sampled wild zebra populations was significantly higher than in zoos suggesting captivity may reduce exposure to EHV-1. Furthermore, the seroprevalence for EHV-1 was significantly higher than for EHV-9 in zebras. In contrast, EHV-9 antibody prevalence was high in captive and wild African rhinoceros species suggesting that they may serve as a reservoir or natural host for EHV-9. Thus, EHV-1 and EHV-9 have a broad host range favoring African herbivores and may have acquired novel natural hosts in ecosystems where wild equids are common and are in close contact with other perissodactyls