In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.
The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific antiLeishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin waxembedded tissues.OF the approximately 20 virulent species of Leishmania, a considerable number has been (Dantas-Torres 2007). Clinical disease in dogs, referred to as canine leishmaniosis (CanL) seems to be caused by only one species, L infantum (syn L chagasi) in many regions worldwide. These protozoal parasites of great medical and veterinary significance are transmitted by the bite of female blood-sucking phlebotomine sand fly vectors of the genera Phlebotomus in the Old World (Desjeux 1996) and Lutzomyia in the New World (Grimaldi and Tesh 1993).Based on serological surveys, it has been estimated that at least 2.5 million dogs are infected in the south-western European countries (Moreno and Alvar 2002). Although most of these dogs are clinically asymptomatic, they constitute a very significant part of the reservoir of L infantum, the aetiological agent of zoonotic visceral leishmaniosis (Dantas-Torres 2007 Frequently, skin biopsies and samples from lymphatic tissue are used for diagnosing CanL. Apart from the microscopic identification of amastigotes, IHC procedures are available. However, commercially available monoclonal antibodies failed to produce specific staining in paraffin wax-embedded tissue and alternatively used canine hyperimmune sera are not readily available for all laboratories and may be cross-reactive with related protozoa (Bourdoiseau and others 1997, Tafuri and others 2004). Thus the major aim of the present study was to fill this diagnostic gap and to establish an in situ hybridisation (ISH) procedure for Leishmania species which facilitates detection of parasites directly within the tissue and which should be specific and sensitive. Material and methods Tissue sam...
In pigs, three different trichomonad species (Tritrichomonas foetus, Tetratrichomonas buttreyi and Tritrichomonas rotunda) have been described as commensals in the large intestine. The aim of this study was to gain further knowledge on the prevalence and pathogenicity of trichomonads in pigs by using a morphology-based approach. Chromogenic in situ hybridization (ISH) is a technique which allows direct localization of the protozoa in the intestinal tissue and correlation of the infection with pathologic changes. In the present study paraffin-wax embedded colon and ileum samples of 192 pigs were analyzed with this method. Using a probe specific for all known members of the order Trichomonadida (OT) 100 of the 192 pigs were tested positive. Thereof, about 10% showed moderate to high-grade parasitic load with trichomonads invading the lamina propria. Partial 18S ribosomal RNA gene sequencing of six of those animals showed a 100% sequence identity with T. foetus sequences. The majority of these animals were also tested positive for other enteropathogenic agents, such as Brachyspira sp., Lawsonia intracellularis, Escherichia coli, and porcine circovirus type 2. All OT-positive samples were further examined with another probe complementary to all known Tritrichomonas species sequences including T. foetus, T. augusta, T. mobilensis and T. nonconforma resulting in only 48 positives. These results suggest that T. foetus may not only be considered as an intestinal commensal but rather a facultative pathogen of pigs with a tendency for tissue invasion in the presence of other agents. Furthermore, the existence of other – yet to be identified – trichomonad species in the colon of pigs was shown.
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.
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