Occurrence of endoparasites in wild Antillean manatees (Trichechus manatus manatus) in Colombia.

The recognized impact of parasites in wildlife populations demands surveillance of endangered species like the Antillean manatees (Trichechus manatus manatus) in Colombia. We conducted a parasitological survey in four rescued sea cows in order to document the parasite diversity of this sirenian in the Caribbean wetland of Colombia and contribute to the molecular characterization of its trematodes. The flukes Chiorchis fabaceus, Nudacotyle undicola and the protozoans Eimeria manatus and E. nodulosa were identified in analysed faecal samples. For C. fabaceus and N. undicola, partial regions of ribosomal RNA genes were amplified and sequenced in order to infer their phylogenetic relations. The current study constitutes a new sirenian host (T. manatus manatus) record for the genus Eimeria and the trematode N. undicola.

Occurrence of anthropozoonotic parasitic infections and faecal microbes in free-ranging sperm whales (Physeter macrocephalus) from the Mediterranean Sea.

Sperm whales (Physeter macrocephalus) are the largest toothed whales and only living member of family Physeteridae. Present survey represents first report on cultivable faecal microbes and gastrointestinal helminths and protozoans infecting free-ranging sperm whales inhabiting Mediterranean Sea waters surrounding Balearic Archipelago, Spain. Twenty-five individual sperm whale scat samples, including one calf, were collected without disturbance of animals during the summer of 2016. Parasitological diagnostic methods, such as sodium acetate acetic formalin (SAF) method, carbol fuchsin-stained faecal smears, Giardia/Cryptosporidium coproantigen ELISAs and an Anisakis-specific PCR were applied for further identification. Five bacterial genera, i.e. Acinetobacter, Clostridium, Enterococcus, Staphylococcus and Streptococcus, and one fungus namely Cladosporium were identified. Parasitological infections included seven different parasite species with some of them bearing anthropozoonotic potential. Thus, four of these parasites were zoonotic, i.e. Anisakis, Balantidium, Diphyllobothriidae gen. sp. and Giardia. Additionally, Zalophotrema curilensis eggs, spirurid-like eggs and Cystoisospora-like oocysts were identified. Molecular characterization identified Anisakis physeteris as the species infecting these whales. This survey provides first records on occurrence of two zoonotic enteropathogenic protozoan parasites (Giardia and Balantidium) and of facultative pathogenic bacteria (Clostridium and Enterococcus) in sperm whales. Presented data should be considered as a baseline study for future monitoring surveys on anthropozoonotic pathogens affecting free-living sperm whale populations and enhance investigations on possible impact on public health as well as on isolated Mediterranean sperm whale subpopulation.

Prevalence of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Crenosoma vulpis larvae in native slug populations in Germany.

Metastrongyloid parasites represent sparsely studied parasites of dogs and cats in Germany. Recent European surveys indicate that these parasites are spreading in Europe. Actual data on prevalence of Angiostrongylus vasorum in dogs and foxes reveal several endemic foci in Germany. However, actual data on the prevalence of A. vasorum and other metastrongyloid lungworm larvae in a wide range of slug and snail intermediate hosts, such as Arion lusitanicus, are missing for Germany. To fill this gap, we conducted an epidemiological survey on native German slugs in selected regions of Hesse and Rhineland-Palatinate. The focus was on slugs, because in study areas slugs appear to be more abundant than snails. Slugs were collected throughout different seasons of the year in areas that were previously proven to be hyperendemic for A. vasorum fox infections. Overall, a total of 2701 slugs were collected and examined for lungworm larvae via artificial digestion. The number of A. vasorum larvae per slug varied considerably (1-546 larvae per specimen). Some hotspot areas with high A. vasorum prevalence in slugs (up to 19.4%) were identified. The overall A. vasorum prevalence varied with season with largest number of slugs infected in summer (9.1%) and lowest number in winter (0.8%). The current study revealed a total A. vasorum prevalence of 4.7% in slugs based on microscopic analyses. Confirmation of lungworm species was made by specific duplex-real-time PCRs. Hence, these data demonstrate that final hosts are at a permanent risk for A. vasorum infections during all seasons when living in investigated areas. Besides A. vasorum, other lungworm larvae were also detected, such as Crenosoma vulpis (the fox lungworm, 2.3%) and Aelurostrongylus abstrusus (feline lungworm, 0.2%).

Regional report on Angiostrongylus vasorum in Colombia: Genetic similarity to European lineage.

The canine lungworm Angiostrongylus vasorum is considered neglected in South America and was only sporadically reported in dogs and wildlife. Gastropods act as obligatory intermediate hosts for this parasitosis. We here analysed Achatina fulica (African giant snail) populations from 5 regions of Colombia for A. vasorum infections. In total, 609 snails were collected from the departments Antioquia, Valle del Cauca and Putumayo. Angiostrongylus vasorum-infected A. fulica were found in all departments with a total prevalence of 3.9%. Larvae originating from Putumayo were molecularly characterized and identified as the European lineage of A. vasorum. This regional report shows for the first time the presence of A. vasorum in intermediate hosts in Colombia and the European genotype in South America.

[Canine peritoneal larval cestodosis caused by Mesocestoides spp. larval stages]. / Kanine peritoneale larvale Zestodose durch Mesocestoides spp.

In a female dog with unspecific clinical symptoms, sonography detected a hyperechoic mass in the middle abdomen and blood analysis a middle grade systemic inflammatory reaction. Laparotomy revealed a peritoneal larval cestodosis (PLC). The diagnosis of an infection with tetrathyridia of Mesocestoides spp. was confirmed by parasitological examination and molecularbiological analysis. Reduction of the intra-abdominal parasitic load as well as a high dose administration of fenbendazole over 3 months led to a successful treatment which could be documented sonographically and by decreased concentrations of C-reactive protein (CRP). Seven months after discontinuation of fenbendazole administration, PLC recurred, pre-empted by an elevation of serum CRP values. According to the literature a life-long fenbendazole treatment was initiated. In cases of unclear chronic granulomatous inflammations in the abdominal cavity in dogs, PLC should be considered. CRP concentration and sonographic examinations are suitable to control for treatment success and a possibly occurring relapse.

Besnoitia besnoiti infections activate primary bovine endothelial cells and promote PMN adhesion and NET formation under physiological flow condition.

Besnoitia besnoiti is an obligate intracellular and emerging coccidian parasite of cattle that mainly infects host endothelial cells during acute infection. We here analyzed early innate immune reactions of B. besnoiti-infected primary bovine umbilical vein endothelial cells (BUVEC). B. besnoiti infections significantly activated BUVEC since the gene transcripts of several adhesion molecules (P-selectin, intercellular adhesion molecule 1(ICAM-1)), chemokines (CXCL1, CXCL8, CCL5), and of COX-2 were significantly upregulated during in vitro infection. Overall, the highest upregulation of most transcripts was observed at 24 or 48 h post infection (p.i.). Enhanced adhesion molecule expression in infected host cells was confirmed by PMN adhesion assays being performed under physiological flow conditions revealing a significantly increased PMN adhesion on B. besnoiti-infected BUVEC layers at 24 h p.i. Furthermore, we were able to illustrate neutrophil extracellular traps (NETs) being released by PMN under physiological flow conditions after adhesion to B. besnoiti-infected BUVEC layers. The present study shows that B. besnoiti infections of primary BUVEC induce a cascade of pro-inflammatory reactions and triggers early innate immune responses.

Gastrointestinal parasite fauna of Emperor Penguins (Aptenodytes forsteri) at the Atka Bay, Antarctica.

In general, the knowledge on parasites infecting Antarctic birds is scarce. The present study intends to extend the knowledge on gastrointestinal parasites of Emperor Penguins (Aptenodytes forsteri) at the Atka Bay, Antarctica. Fecal samples of 50 individual Emperor Penguins were collected at the Atka Bay and analyzed using the sodium-acetate-formaldehyde (SAF) method for the identification of intestinal helminth eggs and/or protozoan parasite stages. In addition, coproantigen ELISAs were performed to detect Cryptosporidium and Giardia infections. Overall, 13 out of 50 penguins proved parasitized (26%). The following stages of gastrointestinal parasites were identified: One Capillaria sp. egg, Tetrabothrius spp. eggs, Diphyllobothrium spp. eggs, and proglottids of the cestode Parorchites zederi. The recorded Capillaria infection represents a new host record for Emperor Penguins. All coproantigen ELISAs for the detection of Cryptosporidium spp. and Giardia spp. were negative. This paper provides current data on parasites of the Emperor Penguin, a protected endemic species of the Antarctica.

Gastrointestinal parasites of free-living Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the Northern Red Sea, Egypt.

The present study represents the first report on the gastrointestinal parasite fauna infecting the free-living and alive Indo-Pacific bottlenose dolphins (Tursiops aduncus) inhabiting waters of the Red Sea at Hurghada, Egypt. A total of 94 individual faecal samples of the examined bottlenose dolphins were collected during several diving expeditions within their natural habitats. Using classical parasitological techniques, such as sodium acetate acetic acid formalin method, carbol fuchsin-stained faecal smears, coproantigen ELISA, PCR and macroscopical analyses, the study revealed infections with 21 different parasite species belonging to protozoans and metazoans with some of them bearing zoonotic and/or pathogenic potential. Four identified parasite species are potential zoonotic species (Giardia spp., Cryptosporidium spp., Diphyllobothrium spp., Ascaridida indet.); three of them are known to have high pathogenic potential for the examined dolphin species (Nasitrema attenuata, Zalophotrema spp. and Pholeter gastrophilus) and some appear to be directly associated with stranding events. In detail, the study indicates stages of ten protozoan species (Giardia spp., Sarcocystis spp., Isospora (like) spp., Cystoisospora (like) spp., Ciliata indet. I and II, Holotricha indet., Dinoflagellata indet., Hexamita (like) spp., Cryptosporidium spp.), seven trematode species (N. attenuata, Nasitrema spp. I and II, Zalophotrema curilensis, Zalophotrema spp., Pholeter gastrophilus, Trematoda indet.), one cestode species (Diphyllobothrium spp.), two nematode species (Ascaridida indet, Capillaria spp.) and one crustacean parasite (Cymothoidae indet.). Additionally, we molecularly identified adult worms of Anisakis typica in individual dolphin vomitus samples by molecular analyses. A. typica is a common parasite of various dolphin species of warmer temperate and tropical waters and has not been attributed as food-borne parasitic zoonoses so far. Overall, these parasitological findings include ten new host records for T. aduncus (i.e. in case of Giardia spp., Sarcocystis spp., Cryptosporidium spp., Nasitrema spp., Zalophotrema spp., Pholeter gastrophilus, A. typica, Capillaria spp., Diphyllobothrium spp. and Cymothoidae indet.). The present results may be used as a baseline for future monitoring studies targeting the impact of climate or other environmental changes on dolphin's health conditions and therefore contribute to the protection of these fascinating marine mammals.

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence.

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n=92; Belo Horizonte, n=50; Nanuque, n=102) and the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=28; Nanuque, n=66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.

In vivo expression profiles of cytokine and iNOS mRNAs in rats infected with Eimeria separata.

Studies on cytokine (IFN-gamma, IL-2, IL-4, IL-5, IL-10) and inducible NO-synthase (iNOS) gene transcription in mesenteric lymph nodes (MLN) and the caecum wall were performed 0, 48, and 72h after primary and challenge infections of rats with Eimeria separata using RT-PCR. The amount of IFN-gamma mRNA was elevated in MLN and caeca 72h after primary and 48-72h after challenge infection when compared with uninfected controls. Increased amounts of IL-2 mRNA were only found in MLN of infected rats 72h post-infection (p.i.). In case of IL-10, infections did not affect the amount of mRNA in MLN, but led to markedly increased levels in the caecum wall of both infected groups 48 and 72h p.i. Levels of IL-4 mRNA remained unchanged after infections and IL-5 gene transcripts were undetectable. Amounts of iNOS mRNA (not investigated in MLN) were found strongly enhanced 48 and 72h p.i. in the caecum walls of all infected animals when compared with naive controls. The data are discussed in regard of the cellular source of the cytokines and their immunological role.

Expression of the microfilarial sheath protein 2 (shp2) of the filarial parasites Litomosoides sigmodontis and Brugia malayi.

The microfilarial sheaths of the filarial parasites Brugia malayi, Brugia pahangi, and Litomosoides sigmodontis consist of several parasite proteins, probably ranging between 7 and 10. The gene encoding sheath protein 2 (shp2), which is the object of this study, is transcribed in embryos and in the uterine epithelium; at least in B. malayi, it is translated in both tissues. Apparently, shp2 is synthesized as a monomer, exported by the respective cells, and integrated into the microfilarial sheath. In the sheath, it exists as a highly polymerized molecule cross-linked by cysteine formation and other covalent bonds, presumably epsilon-(gamma-glutamyl)-lysine links.

Brugia spp. and Litomosoides carinii: identification of a covalently cross-linked microfilarial sheath matrix protein (shp2).

A microfilarial sheath protein gene (shp2) coding for the major constituent of the insoluble, cross-linked sheath remnant (SR) from Brugia malayi, Brugia pahangi and Litomosoides carinii has been cloned and sequenced, based on peptide partial amino-acid sequences. All three closely related single-copy shp2 genes in the two genera carry a single intron in identical position; shp2 mRNAs are post-transcriptionally modified by both cis-splicing and trans-splicing. In accordance with their extracellular destinations the encoded proteins include signal peptide sequences; molecular masses of approx. 23 kDa are hence predicted for the mature secreted polypeptides. In their structures sheath matrix proteins shp2 may be regarded as extreme cases of a modular constitution, since these proteins largely consist of two different segments of multiple sequence repetitions, PAA and QYPQAP (or QYPQ), separated by elements of unique sequence. Extreme insolubility and cross-linking are likely to originate from these repetitive sequences within shp2, and to constitute the basic properties of a microfilarial matrix largely consisting of an shp2 network.

Litomosoides carinii microfilarial sheaths: partial amino acid sequences of several major polypeptide constituents.

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

Trans-splicing of an early embryo mRNA in Litomosoides carinii, coding for the major microfilarial sheath protein gp22.

Both genomic and cDNA clones have been isolated encoding the major sheath glycoprotein, gp22, of Litomosoides carinii microfilariae. The mature gp22 mRNA is shown to result from both trans-splicing of a 22-nucleotide 5'-leader sequence to an acceptor site at position 313 of the pre-mRNA, immediately upstream from the start codon, and from cis-splicing of a 117-nt intron located within the coding sequence. Cis-splicing precedes the trans-splicing reaction. The gp22 reading frame of 148 codons has the inferred structure of a prepro-protein and includes a leader peptide and a pro-segment ahead of the known N terminus of the mature, extracellular protein of 105 amino acids. The N-terminal part of that protein contains five repeats of an elastin-related pentapeptide sequence, which, together with a proline-threonine segment between two Cys clusters in the center and at its C terminus, may cause an elongated conformation with an apparent molecular size of 22 kDa in contrast to the calculated M(r) of 11,200.

Mechanism of catalysis of Fe(II) oxidation by ferritin H chains.

Recombinant H chain ferritins bearing site-directed amino acid substitutions at their ferroxidase centres have been used to study the mechanism of catalysis of Fe(II) oxidation by this protein. UV-difference spectra have been obtained at various times after the aerobic addition of Fe(II) to the recombinants. These indicate that the first product of Fe(II) oxidation by wild type H chain apoferritin is an Fe(III) mu-oxo-bridged dimer. This suggests that fast oxidation is achieved by 2-electron transfer from two Fe(II) to dioxygen. Modelling of Fe(III) dimer binding to human H chain apoferritin shows a solvent-accessible site, which resembles that of ribonucleotide reductase in its ligands. Substitution of these ligands by other amino acids usually prevents dimer formation and leads to greatly reduced Fe(II) oxidation rates.

Ferritins of Schistosoma mansoni: sequence comparison and expression in female and male worms.

Recombinant clones of Schistosoma mansoni cDNA libraries containing the complete coding regions of 2 different ferritin subunits have been isolated and sequenced. This allows for the first time a comparison of ferritin sequences from an invertebrate with those of vertebrates. The deduced amino acid sequences of both Schistosoma ferritin subunit clones show significant homology to vertebrate ferritin H chains. Similarity exceeds 50% identity and includes the recently identified ferroxidase center which is present only in H chains. However, non-conservative substitutions of amino acid residues lining the 3-fold symmetry channel were found, and a gap of 3 successive amino acids unique to the 2 Schistosoma ferritin sequences was identified. Remarkably, for each of the 2 genes, we found a conspicuous difference in the amount of ferritin transcripts between females and males: one of the genes is preferentially expressed in females, the other in males.

Identification and localisation of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni.

An abundant 0.9 kb female-specific mRNA in Schistosoma mansoni is thought to code for an egg-shell precursor protein [Bobek et al. (1986) Proc. Natl. Acad. Sci. USA 83, 5544-5548]. This gene contains two ORFs. A recombinant plasmid was constructed that expresses a fusion protein containing a glycine- and tyrosine-rich polypeptide coded for by one of these ORFs. Antisera raised against homogenates of female, but not of male, S. mansoni recognise this fusion protein, providing direct evidence that this ORF is used by S. mansoni. In comparative Western blots of S. mansoni homogenates from males and females affinity purified antibodies that react with the fusion protein react exclusively with proteins from females, recognising a 28 kDa polypeptide and a smear of immunoreactive material probably caused by oxidative crosslinking. In immunohistology, the affinity purified antibodies react with mature vitelline cells in female schistosomes. The immunoreactive material is localised in the so-called 'vitelline droplets' that are morphologically very similar to 'shell globules', known to contain egg-shell precursors, that are found in Fasciola hepatica. In situ hybridisation shows that the eggshell precursor gene is only transcribed in immature vitelline cells and has a short half-life. Taken together, these observations provide persuasive evidence that the 0.9 kb mRNA codes for an eggshell precursor.