Unusual biphasic disease in domestic pigeons (Columba livia f. domestica) following experimental infection with Sarcocystis calchasi.

A novel Sarcocystis species has recently been reported in the domestic pigeon (Columba livia f. domestica) as intermediate host, causing severe central nervous signs similar to Paramyxovirus-1 or Salmonella Typhimurium var. cop. infection. Transmission of the parasite via the northern goshawk (Accipiter gentilis) as definitive host has been established. Experimental infection of domestic pigeons with sporocysts excreted by experimentally infected northern goshawks reproduced the natural infection in the pigeon, proving the causative role of the parasite in the disease. Here, we describe in greater detail the course of the fulminant biphasic disease depending on the infectious dose. Pigeons infected with 10(3) or 10(4) sporocysts showed clinical signs of polyuria and apathy around 10-11 days postinfection (dpi) and sudden neurological signs 51-57 dpi as a second phase of disease. Pigeons infected with higher doses died within 7-12 dpi, also showing polyuria and apathy but without nervous signs. At necropsy, livers and spleens had multifocal necroses and infestations with parasitic stages, namely, schizonts. Moreover, lesions and schizonts were also found in the lung, bone marrow, and next to blood vessels in the connective tissue of various organs. Pigeons infected with 102 sporocysts remained symptomless until 58-65 dpi, when sudden central nervous signs occurred. Major histopathologic findings of pigeons with neurological signs were encephalitis and myositis of virtually every skeletal muscle with high infestations of sarcocysts. Only mild myocarditis and very few cysts were found in the heart muscles. Importantly, a sentinel pigeon developed identical lesions when compared to those of low-dose infected pigeons, suggesting a risk of mechanical transmission of sporocysts from freshly infected to uninfected pigeons in a flock. By contrast, chickens failed to develop any clinical signs or pathologic lesions in the same experiment. The findings further characterize the new highly pathogenic disease in domestic pigeons, which clinically mimics paramyxovirosis and salmonellosis in both phases of the disease and exclude chickens as further intermediate host species.

A novel Sarcocystis-associated encephalitis and myositis in racing pigeons.

Sarcosporidian cysts in the skeletal muscle of domestic pigeons (Columba livia f. domestica) have previously been attributed to infection with Sarcocystis falcatula, which is shed in the faeces of the opossum (Didelphis virginiana). Here, we describe fatal spontaneous encephalitis and myositis associated with Sarcocystis infections in three flocks of racing pigeons with 47 of 244 animals affected. The clinical course was characterized by depression, mild diarrhoea, torticollis, opisthotonus, paralysis and trembling. Histopathological examination of 13 pigeons revealed generalized severe granulomatous and necrotizing meningoencephalitis and myositis with sarcosporidian cysts. Light and transmission electron microscopy identified cysts in heart and skeletal muscle of 1 to 2 mm in length and 20 to 50 microm in width. These were subdivided into small chambers by fine septae and filled with lancet-shaped cystozoites (7.5 x 1.5 microm) and dividing metrocytes, which is characteristic for Sarcocystis. The cysts had smooth walls and were devoid of protrusions typical of S. falcatula. Polymerase chain reaction amplification and sequencing of the internal transcribed spacer region (ITS-1) and the complete 28S rRNA identified a novel Sarcocystis species with only 51% ITS-1 nucleotide sequence similarity with S. falcatula. A phylogenetic comparison of the 28S rRNA revealed close sequence homologies with Frenkelia microti, Frenkelia glareoli and Sarcocystis neurona. The clinical, histopathological, electron microscopic and genetic data are unlike any previously described protozoan infections in pigeons, suggesting a novel, severe disease due to an as yet undescribed Sarcocystis species.

Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondi in faeces collected from dogs in Germany.

Faecal samples of 24,089 dogs were examined coproscopically in two veterinary laboratories in Germany between March 2001 and October 2004. In 47 dogs, oocysts of 9-14 microm size were found. Their morphology was similar to those of Hammondia heydorni and Neospora caninum. Samples of 28 of these dogs were further examined by inoculation into gerbils: seven isolates induced a specific antibody response against antigens of N. caninum NC-1 tachyzoites. This response suggests that the isolates contained N. caninum. In addition to H. heydorni (12 times isolated), Toxoplasma gondii occysts (twice) and Hammondia hammondi oocysts (twice) were observed in dog faeces. The latter findings suggest that coprophagia with a subsequent intestinal passage by dogs plays a role in the dissemination of coccidian parasites for which cats are definitive hosts. Five of the seven N. caninum (NC-GER2, NC-GER3, NC-GER4, NC-GER5, NC-GER6) and the two T. gondii isolates (TG-dgGER1, TG-dgGER2) were successfully passaged into cell culture and are now available for detailed characterization. In contrast to oocysts of other parasites, N. caninum oocysts were predominantly found between January and April (Fisher exact; P=0.038). In the sera of dogs shedding N. caninum, no reactions against the immunodominant antigens with apparent molecular weights of 19, 29, 30, 33 and 37 kDa of N. caninum tachyzoites were observed 3-5 weeks after shedding. However, the animals recognized a 152-kDa N. caninum antigen. Compared with those identified as H. heydorni, T. gondii or H. hammondi, N. caninum oocyst isolates were significantly smaller in length with the 75th percentiles

Electron microscopic study of in vitro cultures of Hammondia heydorni (Berlin 1996) tachyzoites after passages through dogs, mice, rats, guinea pigs and jirds.

This study describes the fine structure and reproduction of tachyzoites of Hammondia heydorni (isolate Berlin 1996) after in vitro cultivation of stages isolated from serial passages between dogs and various intermediate hosts such as mice, rats, guinea pigs, jirds, knock-out mice, goats and sheep. The tachyzoites are identical with respect to their ultrastructural features to the corresponding stages of the NC-1-strain of Neospora caninum. This fact conforms with other already published data and underlines our opinion that the recently redescribed N. caninum Dubey et al. 2002 is a synonym of the previously described H. heydorni (Tadros and Laarman 1976) Dubey 1977.

Hammondia heydorni-like oocysts shed by a naturally infected dog and Neospora caninum NC-1 cannot be distinguished.

This study describes transmission experiments using Hammondia heydorni-like oocysts isolated in 1996 from a naturally infected dog. The isolate was designated as H. heydorni-Berlin-1996. Examination of sera from infected intermediate hosts showed immunoblot reactions that resembled patterns observed after Neospora caninum NC-1 infection. Furthermore, N. caninum DNA could be demonstrated in tissue samples (e.g. heart, brain) of experimentally infected intermediate hosts and in oocyst preparations from H. heydorni-Berlin-1996. The isolated oocysts did not induce any detectable disease in any of the inoculated adult intermediate hosts (goats, sheep, gerbils, guinea pigs, multimammate rats, BALB/c mice, SCID mice), even upon immunosuppression. Furthermore, neither histological lesions nor parasite stages could be identified in the tissues of all fetuses recovered from two multimammate rats that had been infected prior to pregnancy. An experiment with one dog fed a second time on infected intermediate host tissue indicated that immunity may prevent repeated oocyst shedding in N. caninum-infected dogs. In addition, the study clearly demonstrates that N. caninum can be readily transmitted by dogs that have ingested exclusively skeletal muscles of infected intermediate hosts. Therefore, the study has consequences for the recommendations for farmers to prevent postnatal transmission of N. caninum to cattle. It indicates that feeding of any tissues of potential intermediate hosts (including sheep, goats, rodents) to final hosts may induce the shedding of oocysts in these hosts and thus pose a risk for post-natal infection of cattle. With respect to oocyst morphology and the infectivity of muscle tissues for final hosts, no differences were seen in comparison with observations made in the past on Isospora bigemina/I. heydorni/H. heydorni. Therefore, earlier studies made on I. bigemina/I. heydorni/H. heydorni have to be re-evaluated critically to determine whether they may have included N. caninum or other protozoan parasites that use dogs as final hosts and have an oocyst morphology resembling that of I. bigemina/I. heydorni/H. heydorni.

Cyclic transmission of Neospora caninum: serological findings in dogs shedding oocysts.

In a previous paper we demonstrated that Hammondia heydorni-like oocysts isolated in 1996 from a naturally infected dog could not be distinguished from the isolate Neospora caninum NC-1. The isolate, designated as H. heydorni-Berlin-1996, was cyclically transmitted using dogs as the final hosts. The present study provides information on the antibody responses of the dogs used for the cyclical transmission of this isolate. The majority of dogs that had shed oocysts showed no sero-conversion with respect to N. caninum tachyzoite surface or immunodominant antigens, either in the indirect fluorescent antibody test or in two Western-blot-based tests. In addition to the examination of responses to immunodominant antigens, we also analysed the antibody reactions of dogs to a high-molecular-weight antigen (152 kDa) in the tachyzoite antigen preparation. The antibodies against this antigen appeared after the dogs had been fed infected intermediate host tissues and shed oocysts. The reaction was observed in dogs between day 35 and day 447 after feeding of intermediate host tissues. Therefore, our study provides initial information on a 152 kDa tachyzoite antigen, which might be a suitable candidate to identify dogs with a history of shedding N. caninum oocysts.

Comparative molecular investigation of Nc5-PCR amplicons from Neospora caninum NC-1 and Hammondia heydorni-Berlin-1996.

The clinical relevance of Neospora caninum as a cyst-forming coccidian parasite is increasingly acknowledged within veterinary medicine, although the pathways of transmission are far from being solved. The parasite is well known for causing diaplacental infections in cows associated with abortion and/or severe damage of the fetus. In addition, it may cause neuromuscular disease in dogs, which thus apparently act as intermediate hosts as well as final hosts. In our previous studies, we have demonstrated that molecular diagnosis of N. caninum infections has a high performance when a highly sensitive polymerase chain reaction (PCR) targeted to the Nc5 region of the parasite is used. The present study indicates that the high sensitivity of the PCR is the consequence of a target dose effect which reflects a high redundancy of Nc5-type sequences within the genome of the parasite. The PCR was shown to amplify a set of DNA molecules exhibiting significant sequence differences. A complex composition of Nc5-type sequences was observed in the parasite isolate N. caninum NC-1 but also in another isolate, designated Hammondia heydorni-Berlin-1996. Investigation of the infection pattern of this parasite in its intermediate and final canine hosts showed it to be indistinguishable from N. caninum NC-1.

Further remarks on Hammondia hammondi and the taxonomic importance of obligate heteroxeny.

In this opinion-paper reasons are given why Hammondia hammondi cannot be considered as a separate species, but should be kept as a species of the genus Toxoplasma, if not a strain of Toxoplasma gondii.

Neospora caninum: is it really different from Hammondia heydorni or is it a strain of Toxoplasma gondii? An opinion.

The published data concerning Toxoplasma gondii, Hammondia hammondi, H. heydorni and Neospora caninum on one side and between T. gondii on the other were neglected by most authors. As conclusion we are convinced that there are only two valid species: Isospora (Toxoplasma) gondii and Hammondia heydorni. The first includes as a strain H. hammondi and the latter N. caninum. In any case there is absolutely no reason (with respect to general Zoological nomenclature) to create new genera!

Identification of synapomorphic characters in the genus Sarcocystis based on 18S rDNA sequence comparison.

In order to further investigate synapomorphic characters in the genus Sarcocystis, the small subunit ribosomal RNA gene sequences of Sarcocystis capracanis and Sarcocystis moulei were determined and used to infer the phylogenetic position of these two organisms within the cyst-forming coccidia. Phylogenies derived using distance, maximum parsimony and maximum likelihood methods demonstrated that S. capracanis groups with Sarcocystis tenella and Sarcocystis arieticanis as a clade that shares the characteristic of using canids as their definitive host. S. moulei was shown to group with Sarcocystis gigantea and Sarcocystis fusiformis as a clade that shares the characteristic of using fields as their definitive host.

[Isolation and experimental transmission of Sarcocystis moulei Neveu-Lemaire, 1912]. / Isolation und experimentelle Ubertragung von Sarcocystis moulei Neveu-Lemaire, 1912.

Of 44 several years old goats slaughtered in Kabul/Afghanistan 16 proved to be infected ith Sarcocystis moulei. The large white cysts measuring 2-16 x 1-9 mm were found exclusively in oesophageal muscle tissues of the animals. A total of 4 million sporocysts were harvested from mucosal scrapings of the small intestines of two young cats which had been killed 11 or 14 days after being fed with S. moulei-containing oesophagi of those goats. For transmission experiments in Berlin 11 kids from a S. moulei-free goat breeding as well as 5 Sarcocystis-free cats were used. In a preliminary experiment 3 young goats were orally inoculated with 200 or 2000 sporocysts from Kabul. In further consecutive trials each of the 8 remaining kids received 1000 sporocysts, which were isolated from the intestines of cats after feeding them S. moulei-cysts from successfully infected goats of the first experiment in Berlin. No cysts could be detected in oesophageal muscle tissues of two goats which were slaughtered 4 or 6 months after sporocyst application. Also no sporocysts were excreted by two cats which were fed with the oesophagi of those two animals. In oesophageal muscle tissues of the 9 remaining goats, which were slaughtered 19, 25, 43, 44, 59 or 86 months after infection, 17 to 140 macroscopically visible cysts of S. moulei were found. A definite correlation between age and size of cysts was observed. 19 months p.i. cysts measured 1-2 x 0.5-1 mm, at 43 or 44 months p.i. they were already 1-8 x 1-4 mm, and finally 86 months p.i. they reached a size of 7-13 x 5-8 mm. In H.E. stained histological sections cysts appeared to be subdivided into honeycomb like chambers by very prominent septae. In smaller cysts all chambers were filled with sickle shaped cystozoites, whereas in larger cysts only the chambers at the periphery contained cystozoites. Groups of metrocytes were seen within the cyst groundsubstance at the periphery of cysts indicating further growth, even of very large cysts. Cyst walls measured up to 10 microns in thickness and were composed of a primary and a secondary cyst wall. The thin primary cyst wall was folded irregularly to give cauliflower like protrusions, which were up to 4.4 microns high. The parasitized host cell formed a thick surrounding layer of 6 to 7.5 microns, which was covered by a 2 to 2.5 microns thick layer of connective tissue. All three cats which were fed with S. moulei-cysts in Berlin excreted S. moulei-sporocysts with their faeces. Prepatency was 10 days. A total of 100 sporocysts measured 11.6-13.1 x 8.7-9.4 microns.

A comparison of sporozoite and cyst merozoite surface proteins of Sarcocystis.

Surface labeling of Sarcocystis muris and S. suicanis sporozoites with N-hydroxysuccinimide biotin led to the detection of major membrane proteins with relative molecular weights of 29 and 30 kDa, respectively. Immunoblots of Sarcocystis sporozoite proteins probed with sera from infected hosts or with polyclonal monospecific antibodies generated against membrane antigens of cyst merozoites (noncorresponding stages) showed cross-reactivity between the two developmental stages (cyst merozoites and sporozoites) as well as between the species S. muris and S. suicanis. Two-dimensional gel electrophoresis resulted in the identification of isoforms of the sporozoite membrane antigens, with isoelectric points ranging from pH 4.7 to pH 6.4 for S. muris and from pH 4.7 to pH 5.2 for S. suicanis. The molecular masses, the charge heterogeneity, and the immunological reactivity of the surface proteins of Sarcocystis sporozoites were similar to those of cyst merozoites of both species.

Purification of in vitro excysted Sarcocystis sporozoites by passage through a modified DE 52 anion-exchange column.

A method of purifying Sarcocystis sporozoites following in vitro excystation is described. After passage of sporozoites through a modified DE 52 anion-exchange cellulose column, the mean recovery was 88% for S. capracanis, 71% for S. hircicanis, 77% for S. ovicanis and 74% for S. arieticanis. Most of the sporozoites were motile, and the parasite suspension was free of excystation debris.

DNA measurements and ploidy determination of different stages in the life cycle of Sarcocystis muris.

The DNA contents of different stages within the life cycle of Sarcocystis muris were measured cytophotometrically using DNA-specific Feulgen staining. Stages of gamogony were obtained by the transfer of isolated cyst merozoites into cat kidney-cell cultures. For calculation of absolute DNA contents, the amounts of DNA in the parasites were compared with those in chicken erythrocytes, which are known. The measurements revealed that all investigated stages of S. muris contained haploid DNA except the early zygotes, which were diploid. The further development of the zygotes started with a nuclear division, resulting in two daughter nuclei that again revealed haploid DNA values. The results confirm the existence of zygotic meiosis; thus, a haplo-homophasic life cycle is proposed for the Sarcosporidia.

Besnoitia besnoiti: studies on the definitive host and experimental infections in cattle.

Domestic cats, 11 other species of carnivorous mammals, 6 species of snakes, and white-backed vultures were tested for their possible role as definitive hosts of Benoitia besnoiti by feeding with cystic material from chronically infected bovines. None of the species tested is a definitive host; hence, the life cycle of this parasite remains obscure. In attempts to produce clinical cases of besnoitiosis by experimental infection, bovines were inoculated IV, SC, and IP with cystozoites or tachyzoites. Immunosuppression of the animals was essential for the development of severe cases and skin lesions; cystozoites proved to be more pathogenic than tachyzoites.

Fine structure of Sarcocystis arieticanis Heydorn, 1985 in its intermediate and final hosts (sheep and dog).

The developmental stages of S. arieticanis (sheep-dog-cycle) were studied by means of electron microscopy. After oral infection of the sheep with sporocysts two generations of schizonts occur around days 14 and 27 p.i. Both are morphologically more or less similar and are situated immediately inside the cytoplasm of their host cells, which are endothelial cells. They produce somewhat more than 100 merozoites reaching a size of about 6-8 micron X 2.5 micron. The tissue cysts are characterized by hair-like protrusions of the primary cyst wall. These protrusions (reaching a length of 11 micron at the maximum) do not contain fibrils, are folded over and thus running along the surface. These cysts may then appear in light microscopical sections with a smooth cyst wall. The fine structure of the cyst wall was significantly different from the other sheep sarcosporidia. No differences were found in morphology of the cyst merozoites reaching a size of 15 X 2.5 micron. Inside the dog gamogony occurred in the cells of the lamina propria of the lower part of the small intestine. Gamogony was mostly completed after 20 h p.i. and sporogony after 12-14 days p.i. The fine structure of the stages in the final host was similar to that of other Sarcocystis species.