Justyna Bień

The intestinal microbiota dysbiosis and Clostridium difficile infection: is there a relationship with inflammatory bowel disease?

Gut microbiota is a compilation of microorganisms dwelling in the entire mammalian gastrointestinal tract. They display a symbiotic relationship with the host contributing to its intestinal health and disease. Even a slight fluctuation in this equipoise may be deleterious to the host, leading to many pathological conditions like Clostridium difficile infection or inflammatory bowel disease (IBD). In this review, we focus on the role of microbial dysbiosis in initiation of C. difficile infection and IBD, and we also touch upon the role of specific pathogens, particularly C. difficile, as causative agents of IBD. We also discuss the molecular mechanisms activated by C. difficile that contribute to the development and exacerbation of gastrointestinal disorders.

The first detection of Neospora caninum DNA in the colostrum of infected cows

Limited data is available on the vertical transmission of Neospora caninum via the colostrum. The results of our previous research revealed the presence of N. caninum DNA in the milk of seropositive cows. The aim of the present work is to demonstrate parasite DNA in colostrum samples. A polymerase chain reaction using Np21 and Np6 primers was applied to DNA isolated from the colostrum sediment in order to amplify the corresponding genomic Nc-5 region. The expected 328-bp product was obtained in colostrum samples collected both on the calving day and the day after. This is the first detection of N. caninum DNA in the colostrum of seropositive cows, and these findings implicate the possibility of N. caninum transmission through the colostrum.

The parasitic fauna of the European bison (Bison bonasus) (Linnaeus, 1758) and their impact on the conservation. Part 1 The summarising list of parasites noted

During the current century, 88 species of parasites have been recorded in Bison bonasus. These are 22 species of protozoa (Trypanosoma wrublewskii, T. theileri, Giardia sp., Sarcocystis cruzi, S. hirsuta, S. hominis, S. fusiformis, Neospora caninum, Toxoplasma gondii, Cryptosporidium sp., Eimeria cylindrica, E. subspherica, E. bovis, E. zuernii, E. canadensis, E. ellipsoidalis, E. alabamensis, E. bukidnonensis, E. auburnensis, E. pellita, E. brasiliensis, Babesia divergens), 4 trematodes species (Dicrocoelium dendriticum, Fasciola hepatica, Parafasciolopsis fasciolaemorpha, Paramphistomum cervi), 4 cestodes species (Taenia hydatigena larvae, Moniezia benedeni, M. expansa, Moniezia sp.), 43 nematodes species (Bunostomum trigonocephalum, B. phlebotomum, Chabertia ovina, Oesophagostomum radiatum, O. venulosum, Dictyocaulus filaria, D.viviparus, Nematodirella alcidis, Nematodirus europaeus, N. helvetianus, N. roscidus, N. filicollis, N. spathiger, Cooperia oncophora, C. pectinata, C. punctata, C. surnabada, Haemonchus contortus, Mazamastrongylus dagestanicus, Ostertagia lyrata, O. ostertagi, O. antipini, O. leptospicularis, O. kolchida, O. circumcincta, O. trifurcata, Spiculopteragia boehmi, S. mathevossiani, S. asymmetrica, Trichostrongylus axei, T. askivali, T. capricola, T. vitrinus, Ashworthius sidemi, Onchocerca lienalis, O. gutturosa, Setaria labiatopapillosa, Gongylonema pulchrum, Thelazia gulosa, T. skrjabini, T. rhodesi, Aonchotheca bilobata, Trichuris ovis), 7 mites (Demodex bisonianus, D. bovis, Demodex sp., Chorioptes bovis, Psoroptes equi, P. ovis, Sarcoptes scabiei), 4 Ixodidae ticks (Ixodes ricinus, I. persulcatus, I. hexagonus, Dermacentor reticulatus), 1 Mallophaga species (Bisonicola sedecimdecembrii), 1 Anoplura (Haematopinus eurysternus), and 2 Hippoboscidae flies (Lipoptena cervi, Melophagus ovinus). There are few monoxenous parasites, many typical for cattle and many newly acquired from Cervidae.

First report of Trichinella pseudospiralis in Poland, in red foxes (Vulpes vulpes)

Nematode worms of the genus Trichinella are one of the most widespread zoonotic pathogens. Natural transmission between hosts can only occur through the ingestion of infected meat. To date, two Trichinella species are known to be etiological agents of disease among domestic animals and wildlife in Poland: T. spiralis and T. britovi. In the last decades, since the administration of an oral vaccination against rabies, the red fox population in Poland has increased exponentially. The study area covers the Nowy Targ region: a mountainous area (585–1138 m above the sea) in southern Poland. Of 24 red foxes examined in the study, four were infected with Trichinella isolates: three were identified as T. britovi and one as T. pseudospiralis. The muscle of red foxes infected with T. britovi harboured 2.75, 3.11, 4.4 LPG and with T. pseudospiralis 0.36 LPG. Trichinella larvae were identified at species level by genomic and mitochondrial multiplex PCR, the products of which were sequenced for comparison with other sequences available in GenBank. The sequences obtained from the Polish T. pseudospiralis isolate, deposited in GenBank under the accession numbers JQ809660.1 and JQ809661.1, matched sequences already published in GenBank. Sequence comparison showed a 100% match with the large subunit ribosomal RNA gene of T. pseudospiralis isolate ISS 013, and a 96–95% match with those of T. pseudospiralis isolates ISS 141 and ISS 470. This is the first report of the identification of T. pseudospiralis larvae from red fox in Poland.

Prevalence of antibodies against Neospora caninum in dogs from urban areas in Central Poland

Neospora caninum is a protozoan parasite which causes abortion in cattle as well as reproduction problems and neurological disorders in dogs. To assess the prevalence of the parasite in urban dogs in the Mazovian Voivodeship, Central Poland, serum samples from 257 dogs were analyzed for the presence of specific IgG antibodies. The examined dogs visited three private veterinary clinics located in Warsaw due to control tests, vaccinations, or other reasons not directly connected with neosporosis. Using ELISA and Western blot, antibodies against the parasite were detected in 56 out of 257 dogs, giving a prevalence of 21.7%. A greater prevalence was observed in female dogs than in males, 28% and 17.3%, respectively, and the differences were statistically significant (p < 0.05). There were no significant differences in seroprevalence of Neospora infection within the age groups (p > 0.05). This study indicates the presence of N. caninum in the Mazovian Voivodeship, in dogs which live in urban areas and exposure of these dogs to the parasite. The fact that seropositive dogs had no contact with cattle confirms the important role of dogs in the parasite’s epidemiology.

In vitro isolation and identification of the first Neospora caninum isolate from European bison (Bison bonasus bonasus L.).

Peripheral blood from European bison (Bison bonasus bonasus L.) living in Białowieża Forest, north-east Poland, were investigated for the presence of antibodies to Neospora caninum and isolation of parasite. Out of 23 animals three of them showed a strong positive response to N. caninum (13%). The white blood cells from two positive and two negative bison were loaded on monolayer Vero cells culture. The first viable tachyzoites were detected only in positive samples at days 60 and 70 after incubation. For the purpose of the identification the isolates, tachyzoites were evaluated by PCR and sequence analysis of fragment of the Nc-5 region. The sequences of N. caninum-specific Nc5 region were found to be identical to those of other N. caninum isolates found in the public database. The isolate was subsequently named NC-PolBb1 and NC-PolBb2. This is the first record of in vitro isolation of N. caninum from naturally infected European bison (Bison bonasus bonasus L.) and the first isolate obtained from the peripheral blood.

Colitis Promotes Adaptation of an Intestinal Nematode: A Heligmosomoides Polygyrus Mouse Model System

The precise mechanism of the very effective therapeutic effect of gastrointestinal nematodes on some autoimmune diseases is not clearly understood and is currently being intensively investigated. Treatment with living helminths has been initiated to reverse intestinal immune-mediated diseases in humans. However, little attention has been paid to the phenotype of nematodes in the IBD-affected gut and the consequences of nematode adaptation. In the present study, exposure of Heligmosomoides polygyrus larvae to the changed cytokine milieu of the intestine during colitis reduced inflammation in an experimental model of dextran sulphate sodium (DSS)- induced colitis, but increased nematode establishment in the moderate-responder BALB/c mouse strain. We used mass spectrometry in combination with two-dimensional Western blotting to determine changes in protein expression and changes in nematode antigens recognized by IgG1 in mice with colitis. We show that nematode larvae immunogenicity is changed by colitis as soon as 6 days post-infection; IgG1 did not recognize highly conserved proteins Lev-11 (isoform 1 of tropomyosin α1 chain), actin-4 isoform or FTT-2 isoform a (14-3-3 family) protein. These results indicate that changes in the small intestine provoked by colitis directly influence the nematode proteome. The unrecognized proteins seem to be key antigenic epitopes able to induce protective immune responses. The proteome changes were associated with weak immune recognition and increased larval adaptation and worm growth, altered localization in the intestine and increased survival of males but reduced worm fecundity. In this report, the mechanisms influencing nematode survival and the consequences of changed immunogenicity that reflect the immune response at the site colonized by the parasite in mice with colitis are described. The results are relevant to the use of live parasites to ameliorate IBD.

The parasitic fauna of the European bison (Bison bonasus) (Linnaeus, 1758) and their impact on the conservation. Part 2 The structure and changes over time

During the last century the recorded parasite fauna of Bison bonasus includes 88 species. These are 22 species of protozoa, 4 trematode species, 4 cestode species, 43 nematode species, 7 mites, 4 Ixodidae ticks, 1 Mallophaga species, 1 Anoplura, and 2 Hippoboscidae flies. There are few monoxenous parasites, the majority of parasites are typical for other Bovidae and Cervidae species and many are newly acquired from Cervidae. This is an evident increased trend in the parasite species richness, in both the prevalence and intensity of infections, which is associated with the bison population size, host status (captive breeding or free-ranging) and the possibility of contact with other ruminant species. In light of the changes to parasite species richness during the last decades, special emphasis shall be given to new parasite species reported in European bison, their pathogenicity and potential implications for conservation.

Characterization and differential expression of cathepsin L3 alleles from Fasciola hepatica.

Fasciola hepatica infections cause significant global problems in veterinary and human medicine, including causing huge losses in cattle and sheep production. F. hepatica host infection is a multistage process and flukes express papain-like cysteine proteases, termed cathepsins, which play pivotal roles in virulence through host entry, tissue migration and immune evasion. Expression of these proteases is developmentally regulated. Recent studies indicate that excystment of infective larvae is dependent on cysteine proteases and together FhCL3 and FhCB account for over 80% of total protease activity detectable in newly excysted juvenile (NEJ) fluke. This paper focuses on members of the cathepsin L gene family, specifically those belonging to the CL3 clade. The cDNA of two novel cathepsin L3 proteases--FhCL3-1 and FhCL3-2 were cloned. The mRNA transcript expression levels for these enzymes were significantly different at various time points in life development stages obtained in vitro, from dormant metacercariae to NEJ 24h after excystment. Maximum expression levels were observed in NEJ immediately after excystment. In all stages examined by Real Time PCR, FhCL3-2 was expressed at a higher level compared to FhCL3-1 which was expressed only at very low levels. Western blot and immunohistochemical analysis also indicated higher expression of the FhCL3-2 allele and its secretory nature. The ability of antibody responses from rats and sheep challenged with F. hepatica to recognize recombinant FhCL3-1 and FhCL3-2 was shown to differ. Differences were also confirmed through the use of anti-rFhCL3-1 and anti-rFhCL3-2 sera in Western blot analysis of juvenile excretory/secretory (ES) material separated by 2D electrophoresis. These results indicate analysis of relative expression of parasite virulence factors from different populations is required, as this will likely impact the effectiveness of vaccines based on these antigens.

Detection of specific antibodies anti-Neospora caninum in the fallow deer (Dama dama).

Sera from 335 farmed fallow deer (Dama dama) at the breeding station in Kosewo Górne in the Mazurian Lake District, North-East Poland, were investigated for the presence of antibodies against Neospora caninum. The distribution of age groups was as follow: >4 years - 154 animals; 2 years - 76 animals; 1 year - 105 animals. Ten sera with the optical density exceeding 0.159 absorbance units (i.e., cut-off value) in ELISA test were also analyzed by Western blot. Western blot analysis revealed seroreactivity against immunodominant N. caninum antigens of 37, 25, and 16kDa; however, in some sera additional bands were also visible. This is the first screening studies for antibodies against N. caninum in farmed fallow deer in Poland, in the region where neosporosis was confirmed in cattle and in farmed and free-ranging European red deer (Cervus elaphus).