|Home | About | Journals | Submit | Contact Us | Français|
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 Trichomonas gallinae, Tritrichomonas 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.
The order Trichomonadida belongs to the phylum Parabasala and comprises anaerobic and amitochondrial protists. The order is further subdivided into four families – Monocercomonadidae, Trichomonadidae, Tritrichomonadidae and Trichomitidae (Hampl et al., 2006; Hampl et al., 2007). All trichomonads harbor hydrogenosomes instead of mitochondria and possess up to six flagella. Aside from the Monocercomonadidae all protozoa from this order exhibit an undulating membrane (a motility organelle) and costa or its rudiment (most likely for mechanical support).
Most trichomonads live as symbionts or parasites in the intestine, but there are also several free living species. The order Trichomonadida includes human and veterinary pathogenic species. Trichomonas vaginalis is the most common and best studied trichomonad in human beings and causes trichomonosis, a sexually transmitted infection of the urogenital tract. In veterinary medicine Tritrichomonas foetus, Trichomonas gallinae and Histomonas meleagridis are of high importance. Tritrichomonas foetus is the causative agent of bovine trichomonosis (Parsonson et al., 1976), a sexually transmitted disease leading to uterine infections, abortions and sterility in heifers. The same trichomonad causes colitis in cats (Levy et al., 2003) and can also be found in the colon of pigs (Tachezy et al., 2002). Trichomonas gallinae causes avian trichomonosis and can be detected in the oral cavity, pharynx, esophagus and crop of birds especially of the order Columbiformes (Stabler, 1954). Another parasite found in many gallinaceous birds is Histomonas meleagridis (Tyzzer, 1920) causing a typhlohepatitis known as histomonosis or blackhead disease. Besides these well characterized protozoal agents, there still exist many not well studied trichomonad species. Furthermore, trichomonad infections often do not cause obvious lesions or trichomonads are overlooked in histological slides after hematoxylin and eosin (HE) staining.
Common methods to detect different trichomonad species either involve wet mount preparations followed by cultivation and microscopical examination (Mehlhorn et al., 2009) or PCR analysis and nucleotide sequencing (Gookin et al., 2007). These methods, however, do not provide information concerning pathogenicity of the protozoal agents. Thus, a technique to visualize the organisms directly in tissue sections is desirable. Chromogenic in situ hybridization (ISH) proved to be a robust and reliable method, which enables detection of the respective parasite directly in tissue, in many cases in association with induced lesions (Chvala et al., 2006). This technique had already been successfully applied by Richter et al. (2008) for detection of Monocercomonas sp. and by Liebhart et al. (2006) for three different protozoal agents of the order Trichomonadida. To further study protozoa from this order, an ISH probe with the ability to virtually detect all hitherto known members of the order Trichomonadida (OT probe) seemed to be a useful tool. This probe would allow quick screening of samples, assessment of pathogenicity of the found trichomonads as well as detection of unknown trichomonad species. Here we describe the design and extensive validation of such a probe and its use in chromogenic ISH.
An ISH probe for the detection of all protozoal agents of the order Trichomonadida was designed: order Trichomonadida (OT) probe. First, extensive homology studies using the Sci Ed Central (Scientific & Educational software, Cary, NC, USA) software package were carried out on all available GenBank sequences of the 18S ribosomal RNA (rRNA) gene of protozoa from the order Trichomonadida. A region of strong homology (with a maximum of two nucleotides difference) present in 18S rRNA gene sequences of all trichomonads was chosen as probe sequence. The selected OT probe sequence was 5′-TTG CGG TCG TAG TTC CCC CAG AGC CCA AGA ACT-3′. Subsequently, this sequence was submitted to Basic Local Alignment Search Tool (BLAST; www.ncbi.nlm.nih.gov/blast.cgi) to search against GenBank sequences and exclude unintentional cross-reactivity. The OT probe sequence was sent to Eurofins MWG Operon (Ebersberg, Germany) for probe synthesis and labeling of the 3′ end with digoxigenin. Consequently, ISH was carried out using the newly designed probe on different protozoal cultures and tissue samples.
Chromogenic ISH was performed according to a previously described protocol (Chvala et al., 2006). Briefly, 3 μm paraffin sections were dewaxed and rehydrated. For proteolysis the slides were treated with proteinase K (Roche, Basel, Switzerland) 2.5 μg/ml in Tris-buffered saline for 30 min at 37°C. Afterwards the slides were rinsed in distilled water, dehydrated in alcohol (95% and 100%) and air dried. The slides were incubated overnight at 40°C with a hybridization mixture, 100 μl of which were composed of 50 μl formamide, 20 μl 20x standard sodium citrate (SSC), 10 μl dextran sulfate (50% w/v), 12 μl distilled water, 5 μl boiled herring sperm DNA (50 mg/ml), 2 μl 50x Denhardt's solution and 1 μl OT probe at a concentration of 20 ng/ml. On the second day the slides were washed with decreasing concentrations of SSC (2x SSC, 1x SSC, 0.1x SSC; 10 min each) to remove non-hybridized probe. Afterwards the slides were incubated with anti-digoxigenin-AP Fab fragments (Roche) (1:200) for 1 h at room temperature. Visualization of the hybridized probe was carried out after an additional washing step using the color substrates 5-bromo-4-chloro-3-inodyl phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT) (Roche). Color development was stopped with TE buffer (pH 8.0) after 1 h incubation. The slides were counterstained with hematoxylin and mounted under coverslips using Aquatex (VWR International, Vienna, Austria).
Several protozoal cultures embedded in paraffin wax, displaying representatives of the order Trichomonadida, were used as positive controls for the designed ISH probe. Cultures contained Monocercomonas colubrorum (Richter et al., 2008), Hypotrichomonas acosta, Pentatrichomonas hominis (GenBank accession no. AY349187) (Kleina et al., 2004), Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum (Liebhart et al., 2006), Tritrichomonas foetus (Tachezy et al., 2002), and Tritrichomonas augusta (GenBank accession no. AY055802) (Tachezy et al., 2002) (culture collection of the Department of Parasitology, Faculty of Science, Charles University Prague and of the Clinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna). All protozoal cultures containing an unknown number of parasites were fixed in 7% buffered formalin and embedded in paraffin wax. Prior to embedding all cultures were soaked with rice starch (3.3 mg/ml) for 5 h and centrifuged at 6,000 × g for 10 min to produce a pellet. The pellet was overlaid with agar, hardened at 4°C, carefully removed from the tube by using a spattle and finally embedded in paraffin wax.
Formalin fixed and paraffin embedded tissue samples of different organs from three different animal species were used to analyze the functionality of the designed OT probe directly in tissue. Two samples including tissue from the colon of a pig and the trachea of a pigeon were obtained from the archive of the Institute of Pathology and Forensic Veterinary Medicine. These samples were chosen after examination of hematoxylin and eosin (HE) stained histological sections had revealed protozoa morphologically consistent with trichomonads. Additionally, tissue from the cecum of a turkey experimentally infected with Histomonas meleagridis (Liebhart et al., 2006) was used for ISH.
As negative controls different sections from formalin fixed and paraffin embedded tissues obtained from the archive containing a large variety of different protozoal agents, bacteria, viruses, and fungi were used (Table 1). Additionally, to rule out non-specific hybridization all protozoal culture and tissue samples containing trichomonads were tested using a probe not expected to hybridize with the present protozoa but specifically with Cryptosporidium sp. (Chvala et al., 2006).
PCR followed by gene sequence analysis was carried out on all protozoal and tissue culture samples to ensure sequence identity and integrity. For this purpose, GenBank sequences of the 18S rRNA gene from the protozoal cultures used in this study were aligned, and primers flanking a region which contained at least three nucleotide differences in-between the species were chosen. Afterwards the primers were submitted to BLAST. BLAST analysis revealed sequence similarity only with protozoa from the phylum Parabasala, but not with any relevant bacteria, fungi or viruses. The designed set of sequencing primers with the sequences: forward: 5′-GGT AGG CTA TCA CGG GTA AC-3′ and reverse: 5′-ACT YGC AGA GCT GGA ATT AC-3′ targeted a fragment of 247-249 bp depending on the species amplified.
Prior to PCR DNA was extracted from 10 μm thick sections from formalin fixed and paraffin embedded material. Therefore, the sections were dewaxed in xylene, washed in ethanol and air-dried. Subsequently, the DNA was extracted using the nexttec Clean Column kit (Nexttec, Leverkusen, Germany) according to the manufacturer's instructions. The PCR reaction master mixture of all PCR reactions consisted of 10 μl HotMasterMix (5Prime, Eppendorf, Hamburg, Germany), 0.4 μM of each primer, 2 μl template DNA and distilled water to a total volume of 25 μl. PCR was started with a first heat denaturation step at 94°C for 2 min, followed by 40 cycles of heat denaturation at 94°C for 30 s, primer annealing at 58°C for 30 s and DNA elongation at 72°C for 1 min. Finally, PCR was completed with a DNA elongation step at 72°C for 10 min.
No positive control was used. Negative control was a PCR reaction containing 2 μl of distilled water instead of template DNA. A 10 μl aliquot of each PCR product was analyzed by gel electrophoresis on a 2% Tris acetate-EDTA-agarose gel. The agarose gel was stained with ethidium bromide and bands were visualized with a BioSens SC-Series 710 gel documentation system using the BioSens gel imaging system software (GenXpress Service & Vertrieb GmbH, Wiener Neudorf, Austria). PCR products showing the predicted size were sequenced in both directions according to Bakonyi et al. (2004), except that DNA purification after sequencing PCR was carried out using the DyeEx 2.0 Spin Kit (QIAGEN, Hilden, Germany) instead of precipitation with ethanol. The acquired nucleotide sequences were subjected to BLAST to search against GenBank sequences.
During probe design BLAST analysis of the probe sequence showed an identity to protozoal agents from the order Trichomonadida between 94-100%, which corresponds to a maximum probe-target mismatch of two nucleotides. Two nucleotide mismatches were observed in the sequence of Hypotrichomonas acosta, between two and zero nucleotides difference depending on the strain were detected for Trichomonas vaginalis, Tetratrichomonas gallinarum and Tetratrichomonas sp. One nucleotide probe-target mismatch was found in sequences of Dientamoeba fragilis, Ditrichomonas honigbergii, Hexamastix kirbyi, Hexamastix mitis, Histomonas meleagridis, Monocercomonas colubrorum, Honigbergiella ruminantium, and the sequence ascribed to Pseudotrichomonas keilini that belongs to a free living species of the genus Honigbergiella (Hampl et al., 2007). Additionally, protozoal agents from the phylum Parabasala exclusively found in the hindgut of termites were detected by BLAST analysis showing a sequence similarity of about 88-100%, belonging either to the order Cristamonadida (Joenia sp.) or Trichonymphida (Trichonympha campanula, Trichonympha sp., Hoplonympha sp., Barbulonympha ufalula, Pseudotrichonympha sp. and Euconympha imla).
All trichomonad sequences achieved from tissue (i.e. Tritrichomonas foetus, Trichomonas gallinae, and Histomonas meleagridis) as well as culture samples by PCR and sequence analysis were identical to the respective gene sequences accessible in GenBank and thus confirmed their identity.
In samples from all cultures (Fig. 1) distinct oval to roundish black signals for the respective trichomonad species: Monocercomonas colubrorum (Fig. 1A), Hypotrichomonas acosta (Fig. 1B), Trichomitus batrachorum (Fig. 1C), Pentatrichomonas hominis (Fig. 1D), Tritrichomonas foetus (Fig. 1E), Tritrichomonas augusta (Fig. 1F), Trichomonas gallinae (Fig. 1G) and Tetratrichomonas gallinarum (Fig. 1H) could be detected. All protozoal cells present in the sections of these preparations were labeled by the probe. The trichomonads were surrounded by net-like light blue agar structures. The same reticulate patterns are visible in HE staining (data not shown) of all protozoal culture sections. Therefore, functionality of the newly designed probe was unequivocally proven using the trichomonad culture samples (Table 1).
For confirmation of the broad reactivity spectrum in tissue sections, three different samples were used. In the colon of a pig with a lymphocytic colitis (Fig. 2A) Tritrichomonas foetus were easily discernible with ISH as distinct black signals on the mucosal surface, inside the crypts and even emigrating into the lamina propria (Fig. 2B).
The trachea of a pigeon infected with Trichomonas gallinae showed a severe diphtheroid-necrotizing inflammation at HE staining (Fig. 3A). With ISH abundant numbers of intensely black stained trichomonads were detected in the tissue which even migrated from the esophagus into deep layers of connective tissue towards the trachea (Fig. 3B). Additionally, cloudy not well delineated black signals were visible especially in the necrotic tissue.
The cecum of a turkey experimentally infected with Histomonas meleagridis showed a severe necrotizing typhlitis and polygonal pale parasite-like objects in the HE stained sections (Fig. 4A). ISH revealed large numbers of distinctly black stained histomonads (Fig. 4B), which corresponded to the pale parasite-like objects observed by histological examination.
In all tested tissue samples no unspecific background staining was present (Table 1).
None of the samples containing organisms or pathogens other than Trichomonadida showed positive reactivity with the OT probe (Table 1). Also, no unspecific reactivity was present in any of the tissue and protozoal culture samples analyzed with the Cryptosporidium probe (Chvala et al., 2006).
This work describes a new ISH application for the detection of all hitherto known protozoal agents of the order Trichomonadida. The OT probe presented here facilitates the detection of various trichomonads especially in tissue samples. All tested cross reactivities of numerous common protozoal agents, fungi, bacteria and viruses displayed negative results. In BLAST analysis of the OT probe not only protists from the order Trichomonadida but also protozoal species of other orders (Cristamonadida and Trichonymphida), only found in the hindgut of termites, were detected. This is likely due to the fact that these protozoa also belong to the phylum Parabasala (Noda et al., 2009) and hence are close relatives. The probable cross reactivity is not expected to cause any problems in veterinary diagnostics, because these termite parasites don't colonize the same host spectrum as trichomonads from vertebrates. Therefore, the presented ISH may be considered a tool for specific detection of parasites belonging to the order Trichomonadida in diagnostic tissue samples.
The functionality of the probe was shown using different protozoal cultures. The observed light blue net-like structures surrounding the distinctly dark blue stained trichomonads are most likely composed of agar flocks. This pattern had an amorphous appearance and thus differed clearly from the uniform oval to roundish shape of the protozoa. The same structures could be seen in HE stained protozoal culture sections. In all three different tested tissues sections three important representatives of trichomonads showed distinct specific labeling. No background was observed only in the tissue sections containing Trichomonas gallinae cloudy, not well delineated signals were visible in severely necrotic areas. Taking into account that non-specific hybridization was excluded by using a non-cross reactive probe, these signals are most likely due to cellular fragments from lysed parasites. Therefore, the specificity and functionality of the probe in protozoal culture and tissue sections was proven.
In routine diagnosis HE staining is commonly used. For trichomonads this staining is suboptimal, based on the fact that the homogeneously eosinophilic trichomonads are often overlooked, especially in necrotic tissue, and in the intestinal tract are often mistaken for cell debris or ingesta. By chromogenic ISH it is easy to detect the trichomonads' rRNA gene due to the intense black to purple staining. Other commonly used detection techniques are either PCR (Grabensteiner and Hess, 2006; Gookin et al., 2007) or parasite cultivation (Gookin et al., 2003) mostly followed either by sequencing or ultrastructural studies for species identification (Rivera et al., 2008; Mehlhorn et al., 2009). Both methods have the advantage that detection can be carried out on fecal samples from living animals, whereas ISH is performed post mortem on tissue sections. However, a problem often observed in parasite cultivation is that some trichomonad species are difficult to cultivate (Crotti et al., 2007) and hence, are not present after cultivation. Even if PCR is used on different tissue samples to study the distribution of protozoa, PCR and parasite cultivation from fecal samples generally have the disadvantage that they do not allow unequivocal interpretation of whether a detected protozoal agent is responsible for clinical signs or pathologic lesions. In contrast, ISH allows visualization of the parasites in the tissue in direct context with accompanying lesions.
In summary, the here described chromogenic ISH is a technique with the main advantage of directly localizing the protozoal agent in the tissue, associating the trichomonads with accompanying lesions as well as estimating the parasitic load and therefore allowing for assessment of pathogenicity but not for species identification. The OT ISH probe described here proved to be a useful tool for broad screening, due to the large variety of different trichomonads, which can be detected. Furthermore, considering the wide diagnostic spectrum of the OT probe, it paves the way for discovering hitherto unknown trichomonad species and it can be seen as the starting point for the design of additional more species-specific probes.
The authors wish to thank Karin Fragner and Klaus Bittermann for excellent technical support. This work was funded by the FWF grant P20926.
Conflict interest statement
The authors declare no conflicts of interest