Design and validation of an oligonucleotide probe for the detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization

Abstract

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 T. gallinae, T. 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.

Loop-mediated isothermal amplification assay for detection ofHistomonas meleagridisinfection in chickens targeting the 18S rRNA sequences

“…It has been successfully introduced to develop PCR, nested PCR, real-time PCR and in situ hybridization for detection of H. meleagridis (Hafez et al, 2005;Huber et al, 2005;Liebhart et al, 2006;Bleyen et al, 2007;Mostegl et al, 2010). In this study, the LAMP primer sets were designed in the conserved regions of the 18S rRNA gene, and results implied that this LAMP is an appropriate tool for detection and differentiation of H. meleagridis from other related parasites.…”

“…Frequent appearance of moderate gross lesions in these organs may be confused with other avian diseases such as Marek's disease, avian leukosis, tuberculosis, mycosis, avian vibrionic hepatitis and coccidiosis (McDougald, 2005). Although H. meleagridis may be further confirmed by direct microscopical observation or certain tissue staining techniques in scrapings of mucosa and cecal droppings, it is difficult to differentiate it from other cecal parasites such as Blastocytis sp and Tetratrichomonas gallinarum (McDougald, 2005;Mostegl et al, 2010). The isolation and stable cultivation of H. meleagridis in vitro have been reported Gerhold et al, 2010;Ganas et al, 2012), but the procedure is still laborious and time-consuming.…”

“…The isolation and stable cultivation of H. meleagridis in vitro have been reported Gerhold et al, 2010;Ganas et al, 2012), but the procedure is still laborious and time-consuming. With the developments of sensitive and specific molecular techniques, conventional polymerase chain reaction (PCR), nested PCR, real-time PCR and in situ hybridization targeting the 18S rRNA gene have been successfully developed to detect the DNA of H. meleagridis in organ samples or faeces in recent years (Hafez et al, 2005;Huber et al, 2005;Liebhart et al, 2006;Bleyen et al, 2007;Mostegl et al, 2010). However, these methods require skillful techniques, long reaction times, expensive and high-precision instruments, which may not be readily available in rural endemic regions.…”

First report of typhlitis/typhlohepatitis caused byTetratrichomonas gallinarumin three duck species

“…A culture of T. gallinarum isolated from the caecal contents of turkeys was used as positive control (Liebhart et al , 2006). Cross-reactivity was further checked directly on various embedded cultures or tissue samples including several species of flagellates, other protozoan parasites, bacteria, fungi and viruses as listed in Mostegl et al (2010) except for T. gallinarum .…”

Development of a chromogenic in situ hybridization for Giardia duodenalis and its application in canine, feline, and porcine intestinal tissues samples

“…Chromogenic ISH, on the other hand, combines specific identification of the infectious agent with the possibility for simultaneous evaluation of associated tissue lesions. The technique has been successfully used for the demonstration of various protozoal parasites in pathology specimens, such as Histomonas meleagridis , Entamoeba sp., Cryptosporidium sp., members of the order Trichomonadida , and others 6,15,20,24. In particular, Giardia trophozoites are difficult to identify in conventionally stained histological slides, as they may easily be confounded with cell debris, secretion products, or ingesta.…”