Morphological and ultrastructural characteristics of Myxobolus ridibundae n. sp. (Myxosporea: Bivalvulida) infecting the testicular tissue of the marsh frog Rana ridibunda (Amphibia: Ranidae) in Egypt.

Myxozoans are one of the most economically important groups of protozoan parasites causing many serious diseases of their hosts. In the present study, a total of 60 live adult male specimens of the marsh frog Rana ridibunda have been randomly captured during the period of January-December 2015 in different areas at Kafr El-Sheikh Governorate, Egypt and were examined for infection by myxosporidian parasites. A total of 48 (80.0 %) out of 60 frog specimens were found to be infected with Myxobolus species. Parasitic infection was restricted to the testicular tissue of the examined frogs. Macroscopic cysts (plasmodia) which heavily infested different parts of the testes were recovered. Morphological and ultrastructural characteristics of these myxosporidian species were carried out using light and transmission electron microscopy. Plasmodia measured 0.16-0.53 (0.34 ± 0.01) mm in diameter. Mature spores appeared oval in frontal view, measuring 8.9-11.5 (9.6 ± 0.1) µm in length and 7.5-9.1 (8.4 ± 0.1) µm in width containing 5-6 turns of polar filaments. Morphometric characterization revealed that the very small size of the present Myxobolus species was the most distinctive feature that separates them from all previously described Myxobolus species. Ultrastructural analysis showed that the plasmodia are surrounded by a plasma membrane with numerous pinocytotic protrusions extending toward the host cell. The generative cells and the different developmental stages are arranged at the periphery of the plasmodia, while immature and mature spores are centrally located. Sporogenesis, capsulogenesis, valvogenesis, and spore maturation of the present parasite are also described. The present species is described as Myxobolus ridibundae and represents a new species.

Morphological re-description and molecular characterization of Kudoa pagrusi (Myxosporea: Multivalvulida) infecting the heart muscles of the common sea bream fish Pagrus pagrus (Perciformes: Sparidae) from the Red Sea, Egypt.

In the present study, 100 samples of different sizes of the common sea bream fish Pagrus pagrus were collected from the Egyptian water along the Gulf of Suez, Red Sea and examined for the prevalence of myxosporidian parasites in general and Kudoa spp. in particular. Fish samples were thoroughly externally examined. After dissection, all the internal organs were removed and examined. A total of 60 out of 100 fish specimens were found to be infected with Kudoa stages. Parasitic infection was restricted to the heart muscles of the examined fish. None of the other organs was found to be infected. Macroscopic cysts (plasmodia) heavily infested the different parts of the heart muscles. Each plasmodium measured 1.2-2.5 (1.53 ± 0.2) mm × 0.63-0.80 (0.65 ± 0.2) mm. Mature spores are quadratic in shape in the apical view showing four equal valves and four symmetrical polar capsules. Fresh spores were 5.0-7.1 (5.7 ± 0.2) µm long × 5.4-8.5 (6.1 ± 0.3) µm wide. On the basis of spore morphology, the present species was identified as Kudoa pagrusi. Morphometric characterization revealed that the relatively small size of this Kudoa species was the distinctive feature that separates it from all previously described species. Molecular analysis based on small subunit ribosomal DNA (SSU rDNA) sequences revealed that the highest percentage of identity was observed with K. scomberomori and followed by K. shiomitsui, K. hypoepicarclialis, K. amamiensis, and K. kenti. The kudoid spores showed morphometric variations to some extents but had essentially identical nucleotide sequences of the SSU rDNA gene sequences closest to those of K. scomberomori and K. shiomitsui recorded from elasmobranchs in the Indo-Pacific Ocean. The present findings support the identification of an ancestral marine origin of the present Kudoa species.

Mosquito vectors and the spread of cancer: an overlooked connection?

Mosquitoes (Diptera: Culicidae) represent a key threat for millions of humans and animals worldwide, vectoring important pathogens and parasites, including malaria, dengue, filariasis, and Zika virus. Besides mosquito-borne diseases, cancers figure among the leading causes of mortality worldwide. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades. Notably, there are few contrasting evidences of the relationship between cancer and mosquito-borne diseases, with special reference to malaria. However, analogies at the cellular level for the two diseases were reported. Recently, a significant association of malaria incidence with all cancer mortality in 50 USA states was highlighted and may be explained by the ability of Plasmodium to induce suppression of the immune system. However, it was hypothesized that Anopheles vectors may transmit obscure viruses linked with cancer development. The possible activation of cancer pathways by mosquito feeding events is not rare. For instance, the hamster reticulum cell sarcoma can be transmitted through the bites of Aedes aegypti by a transfer of tumor cells. Furthermore, mosquito bites may influence human metabolic pathways following different mechanisms, leading to other viral infections and/or oncogenesis. Hypersensitivity to mosquito bites is routed by a unique pathogenic mechanism linking Epstein-Barr virus infection, allergy, and oncogenesis. During dengue virus infection, high viral titers, macrophage infiltration, and tumor necrosis factor alpha production in the local tissues are the three key important events that lead to hemorrhage. Overall, basic epidemiological knowledge on the relationships occurring between mosquito vector activity and the spread of cancer is urgently needed, as well as detailed information about the ability of Culicidae to transfer viruses or tumor cells among hosts over time. Current evidences on nanodrugs with multipotency against mosquito-borne diseases and cancers are reviewed, with peculiar attention to their mechanisms of action.

Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control.

The fight against mosquito-borne diseases is a challenge of huge public health importance. To our mind, 2015 was an extraordinary year for malaria control, due to three hot news: the Nobel Prize to Youyou Tu for the discovery of artemisinin, the development of the first vaccine against Plasmodium falciparum malaria [i.e. RTS,S/AS01 (RTS,S)], and the fall of malaria infection rates worldwide, with special reference to sub-Saharan Africa. However, there are major challenges that still deserve attention, in order to boost malaria prevention and control. Indeed, parasite strains resistant to artemisinin have been detected, and RTS,S vaccine does not offer protection against Plasmodium vivax malaria, which predominates in many countries outside of Africa. Furthermore, the recent outbreaks of Zika virus infections, occurring in South America, Central America and the Caribbean, represent the most recent of four arrivals of important arboviruses in the Western Hemisphere, over the last 20 years. Zika virus follows dengue (which slyly arrived in the hemisphere over decades and became more aggressive in the 1990s), West Nile virus (emerged in 1999) and chikungunya (emerged in 2013). Notably, there are no specific treatments for these arboviruses. The emerging scenario highlights that the effective and eco-friendly control of mosquito vectors, with special reference to highly invasive species such as Aedes aegypti and Aedes albopictus, is crucial. The concrete potential of screening plant species as sources of metabolites for parasitological purposes is worthy of attention, as elucidated by the Y. Tu's example. Notably, plant-borne molecules are often effective at few parts per million against Aedes, Ochlerotatus, Anopheles and Culex young instars, can be used for the rapid synthesis of mosquitocidal nanoformulations and even employed to prepare cheap repellents with low human toxicity. In addition, behaviour-based control tools relying to the employ of sound traps and the manipulation of swarming behaviour (i.e. "lure and kill" approach) are discussed. The importance of further research on the chemical cues routing mosquito swarming and mating dynamics is highlighted. Besides radiation, transgenic and symbiont-based mosquito control approaches, an effective option may be the employ of biological control agents of mosquito young instars, in the presence of ultra-low quantities of nanoformulated botanicals, which boost their predation rates.

Cytokine signature and antibody-mediated response against fresh and attenuated Anisakis simplex (L3) administration into Wistar rats: implication for anti-allergic reaction.

The third larval stage (L3) of Anisakis simplex (Anisakidae) is one of the zoonotic parasitic nematodes in the musculature and visceral organs of marine fishes belonging to family Moronidae. The consumption of these high-commercial-value fish is widespread in many countries around the Mediterranean Sea including Egypt. The presence of these larvae in fish muscles poses a potential consumer hazard due to the parasite's ability to cause anisakidosis. Forty-two out of 60 (70%) of the European seabass Dicentrarchus labrax were found to be naturally infected by L3 of A. simplex in the form of encapsulated juveniles in the fish musculature. Morphological examination of recovered parasites by light and scanning electron microscopy showed that, in general, all specimens examined closely resembled A. simplex (L3). To evaluate the allergenicity of this nematode, white blood cell count; levels of T helper 1 (Th1) [interferon (IFN)-γ and tumor necrosis factor (TNF)-α)], Th2 [IL-4, IL-5, and IL-6], and Th17 [IL-17] related cytokines; total IgE and IgG antibodies; and nitric oxide (NO) were measured in the plasma of Wistar rats sensitized by oral inoculation with fresh, frozen, and heat-treated A. simplex L3 or rats intraperitoneally injected with L3 crude extract. Rats sensitized with fresh and frozen L3 larvae produced significantly higher levels of IFN-γ, IL-5, IL-17, and total IgE as compared to control rats. Heat-treated larvae administration resulted in a significant rise of IFN-γ, TNF-α, IL-5, and total IgE in comparison to control rats. Intraperitoneal sensitizations enhanced release of IFN-γ, TNF-α, and total IgE. Oral sensitization led to a significant production of NO. Thereby, frozen or cooked larval L3 cannot inhibit the release of Th-related cytokines and IgE, which might impact on the overall anti-parasitic immunity.

Discovery of microchannels in host tissues surrounding hydatid cysts.

The present study shows for the first time long, tiny microchannels starting from the surface of a Echinococcus granulosus hydatid and running through the laminated layer and the pericyst into the healthy tissue. It is, however, not clear, whether these channels are persistent or only formed for a short period and whatever function they fulfill.

Integrated fly management in European ruminant operations from the perspective of directive 2009/128/EC on sustainable use of pesticides.

Integrated pest management (IPM) in agriculture animals remains undeveloped as compared to IPM in crops. With respect to the range of external nuisance arthropods that may bother farm animals, development and implementation of systematic IPM strategies are difficult to carry out. However, recurrent outbreaks of blue tongue disease in sheep and cattle, the public threats regarding the prophylactic use of veterinary insecticides and the need to preserve the efficacy of available actives have to lead the reflexion on new control strategies for arthropod pests of livestock. A recent extension of EU regulation on the use of pesticides in crops provides an opportunity to compare IPM strategies and to suggest new lines of reflection for the control of nuisance pests in ruminants under European conditions. In this paper, actions suggested by the Annex III of the Directive 2009/128/CE on Sustainable Use of Pesticides and related National Action Plans from 28 member states of the EU were reviewed from an animal production perspective by a group of veterinary entomologists. Eight lines of action have been identified and thus challenged with respect to current husbandry practices in modern European ruminant operations. Many IPM strategies for crops were identified to be unsuitable for large animals. Suggestions for implementing tools, opportunities and constraint assessment, and needs for support were also discussed. Only control of pest development sites and monitoring of harmful organisms were considered achievable in the near future; both in conjunction with the use of topical insecticides. Complementary actions such as alternatives to chemical control require further researches and industrial development. Marketing of IGR-based feed additives would be of great interest, but development of new compounds for veterinary medicines is very unlikely with respect to the European regulatory environment and associated cost of development.

Sarcocystis arieticanis (Apicomplexa: Sarcocystidae) infecting the heart muscles of the domestic sheep, Ovis aries (Artiodactyla: Bovidae), from K. S. A. on the basis of light and electron microscopic data.

In the present study, the heteroxenous life cycle of Sarcocystis species from three strains of the slaughtered sheep at Al-Azizia and Al-Saada abattoirs in Riyadh city, K.S.A., was studied. Muscle samples of the oesophagus, diaphragm, tongue, skeletal and heart muscles were examined. Varied natural infection rates in the muscles of the examined sheep strains were recorded as 83% in Niemy, 81.5% in Najdy and 90% in Sawakny sheep. Muscles of the diaphragm showed the highest infection level above all organs except Najdy sheep in which oesophagus has the highest rate. Also, the heart was the lowest infected organ (40% Niemy, 44% Najdy and 53% Sawakny). Microscopic sarcocysts of Sarcocystis arieticanis are easily identified in sections through the heart muscles of the domestic sheep Ovis aries (Artiodactyla: Bovidae). Cysts measured 38.5-64.4 µm (averaged 42.66 µm) in width and 62.4-173.6 µm (averaged 82.14 µm) in length. The validity of this species was confirmed by means of ultrastructural characteristics of the primary cyst wall (0.1-0.27 µm thick) which revealed the presence of irregularly shaped crowded and hairy-like projections underlined by a thin layer of ground substance. This layer consisted mainly of fine, dense homogenous granules enclosing the developing metrocytes and merozoites that usually contain nearly all the structures of the apical complex and fill the interior cavity of the cyst. Several septa derived from the ground substance divided the cyst into compartments. The merozoites were banana-shaped and measured 12-16 µm in length with centrally or posteriorly located nuclei. Experimental infection of carnivores by feeding heavily infected sheep muscles revealed that the dog, Canis familiaris, is the only final host of the present Sarcocystis species. Gamogony, sporogonic stages and characteristics of sporulated oocysts were also investigated.

Sarcocystis acanthocolubri sp. n. infecting three lizard species of the genus Acanthodactylus and the problem of host specificity. Light and electron microscopic study.

In the present investigation, macroscopic sarcocysts of Sarcocystis acanthocolubri were observed in muscles of 42 (4.3%) out of 975 Acanthodactylus sp. lizards collected from different geographical areas in Egypt. The infection rate was 6.4% in Acanthodactylus boskianus, 2.1% in Acanthodactylus sculentus, and 5% in Acanthodactylus paradalis. The highest infection rate was recorded in the lizards captured from Baltem (10% in A. boskianus and 8% in A. paradalis). The infection rate was usually higher in females (7.4%) than in males (3.8%). Moreover, the highest infection rate was recorded in summer (7.53%), autumn (3.57%), and spring (3.11%), and the lowest was recorded in winter (0.91%). Also, old animals had higher infection rates (10.8%) than young ones (0-2.7%). Macrocysts measured 0.95 × 10.12 mm. Both macroscopic and microscopic sarcocysts were enclosed only by a primary cyst wall, which had many finger-like, stalkless, and non-branched protrusions giving it a striated appearance. The primary cyst wall measured 3.9 µm. A dark granulated ground substance was found directly underneath the protrusions and is extended interiorly dividing the cyst cavity into many compartments containing the parasites (metrocytes and merozoites). Metrocytes were found directly under the ground substance and usually multiply asexually by endodyogeny producing two merozoites from each metrocyte. Both metrocytes and merozoites had the apical complex structures characteristic to the genus Sarcocystis. Transmission experiments with three snake species indicated that the snake Spalerosophis diadema is the proper final host belonging to the family Colubridae. The prepatent period was 16 days, while the patent period was 35 days. The results obtained from the present investigation revealed that this is a new species which was named Sarcocystis acanthocolubri.

Morphological and molecular biological characterization of Pleistophora aegyptiaca sp. nov. infecting the Red Sea fish Saurida tumbil.

One hundred three out of 225 (45.8%) of the Red Sea fish Saurida tumbil were infected with microsporidian parasites. The infection was recorded as tumor-like masses (whitish macroscopic cysts) or xenomas often up to 2 cm in diameter and embedded in the peritoneal cavity. Generally, the infection was increased during winter 63.8% (86 out of 135) and fall to 18.9% (17 out of 90) in summer. Light microscopic study revealed that xenomas were encapsulated by a fibrous layer encircling numerous sporophorous vesicles filled with mature spores measuring 1.7 ± 0.6 (1.5-2.7 µm) × 1.5 ± 0.3 µm (1.2-1.8 µm) in size. Ultrastructural microscopic study showed the presence of smooth membranes of the sarcoplasmic reticulum forming a thick, amorphous coat surrounding various developmental stages of the parasite. The various recognizable stages of the parasite were uninuclear, binucleated, and multinucleated meronts followed by detachment of the plasmalemma of the sporont from the sporophorous vesicle producing sporoblasts. Mature spores consist of a spore coat and spore contents. The spore contents consist of the uninucleated sporoplasm and a posterior vacuole located at the posterior end. The polar tube consists of a straight shaft and a coiled region (26-32 coils) arranged in many rows along the inside periphery of the spore. The polaroplast consisted of an anterior region of closely and loosely packed membranes. Molecular analysis based on the small subunit rDNA gene was performed to determine the phylogenetic position of the present species. The percentage identity between this species and a range of other microsporidia predominantly from aquatic hosts demonstrated a high degree of similarity (>92%) with eight Pleistophora species. Comparison of the nucleotide sequences and divergence showed that the sequence of the present microsporidium was most similar to that of Pleistophora anguillarum (99.8% identity) differing in 13 nucleotide positions. So, the present species was recorded and phylogenetically positioned as a new species of Pleistophora.

Molecular pathology, taxonomy and epidemiology of Besnoitia species (Protozoa: Sarcocystidae).

Until recently, besnoitiosis has been a neglected disease of domestic animals. Now, a geographic expansion of the causing protozoan parasite Besnoitia besnoiti in livestock has been recognized and the disease in cattle is considered emerging in Europe. Bovine besnoitiosis leads to significant economic losses by a decline in milk production, sterility, transient or permanent infertility of bulls, skin lesions and increase of mortality in affected cattle population. Phylogenetically, the Besnoitia genus is closest related to the well studied and medically important protozoans, Toxoplasma gondii and Neospora caninum. In contrast, discriminative molecular markers to type and subtype large mammalian Besnoitia species (B. besnoiti, B. caprae, B. tarandi, B. bennetti) on a relevant level of species and strains are lacking. Similarly, these cyst-forming parasites may use two hosts to fulfill their life cycle, but this has not been proven for all large mammalian Besnoitia species yet. Most important though, the final hosts and transmission routes of these Besnoitia species remain mysterious. Here, we review aspects of parasite's pathology, speciation, phylogeny, epidemiology and transmission with a special focus on recent molecular studies of all to date known Besnoitia species. Using an integrated approach, we have tried to highlight some promising directions for future research.

Prevalence pattern and biology of Sarcocystis capracanis infection in the Egyptian goats: a light and ultrastructural study.

Cysts of Sarcocystis capracanis obtained from infected goats were examined to clarify the effect of the parasite on the host. Muscle tissues from fresh oesophagus, tongue, diaphragm and skeletal muscles of 680 goats were slaughtered in the main abattoir of Cairo, Egypt and they were examined microscopically for Sarcocystis infection for the first time in Egypt. 540 out of 680 (79.4%) of examined goats were found to be infected with Sarcocystis sp. The infection was recorded firstly by light microscopy as spindle shaped cysts embedded in the muscle tissues. The validity of this species as S. capracanis was confirmed by means of ultrastructural characteristics of the primary cyst wall which revealed the presence of thick-radially striated wall with finger like projections, underlined by a thick layer of ground substance enclosing the developing metrocytes and merozoites that usually contain nearly all the structures of the apical complex and fill the interior cavity of the cyst. The cyst cavity is divided by many septa extending from the ground substance and producing large number of chambers. An experimental infection using the highly infected muscles was carried out to determine the final host, which is dog. Smears of intestinal epithelium were taken to examine the endogenous stages (gamogony and sporogony) by means of light microscopy. These stages were mainly observed as to infect the lamina propria of the posterior third of the small intestine. Gamogony and zygote formation (fertilization) occurred 2-8 days post infection, while sporulation took place within the final host 13-15 days and sporocysts were passed within faeces of the infected puppies at that time. The prepatent period of S. capracanis was 12-15 days, while the patent period was extended to 37 days. In goats, infection with S. capracanis led to the loss of weight, anaemia, abortion and even death in cases of heavy infection. While bleeding, watery faeces filled with mucous on 5th and 8th day p.i. as well as intestinal lesions are the pathogenic effects occurred in puppies after experimental infection.

Microsporidian parasites: a danger facing marine fishes of the Red Sea.

Out of 600 marine fish from the Red Sea belonging to three different species that were collected and examined for microsporidian parasites, 87 (14.5%) fish were found to be infected. The infection was recorded as cysts or xenomas embedded in the gut epithelium and the peritoneal cavity of the three fish species. The highest percent of infection with microsporidian parasites was recorded in Saurida tumbil 19.5% (39/200) followed by Pagrus pagrus 15% (45/300) and the lowest percent of infection was recorded in Epinephelus chlorostigma 3% (three out of 100). After rupture of the cysts, the spores were released and examined by light microscopy. Each spore was elongated to ellipsoidal in shape and possessed a posterior vacuole which is characteristic to phylum Microspora. They measure 1.6 ± 0.5 µm (1.5-2.4 µm) × 1.3 ± 0.1 µm (1.3-2.0 µm) in Saurida tumbil and Pagrus pagrus, respectively. The spores of Pleistophora sp recorded from E. chlorostigma were ovoid to pyriform in shape and measure 1.9 ± 0.5 µm (1.8-2.7 µm) × 1.6 ± 0.4 µm (1.5-2.4 µm).

Sarcocystis calchasi is distinct to Sarcocystis columbae sp. nov. from the wood pigeon (Columba palumbus) and Sarcocystis sp. from the sparrowhawk (Accipiter nisus).

Sarcocystis calchasi has been identified as causative agent of a newly discovered central nervous disease in domestic pigeons (Columba livia f. domestica) observed for the first time in Germany in 2006. Initial studies have indicated that this parasite is highly pathogenic for domestic pigeons after ingestion of low doses of sporocysts shed by the Northern goshawk (Accipiter gentilis). Here we tested whether phylogenetically related birds might regularly harbor Sarcocystis species. Five wood pigeons (Columba palumbus) and five sparrowhawks (Accipiter nisus) from Northern Germany were examined. All birds were PCR negative for S. calchasi by universal primers. Instead, both avian species harbored two as yet undescribed Sarcocystis species. Light and transmission electron microscopy identified cysts in the skeletal muscle of wood pigeons of 56-156 microm in width. The cysts had a smooth surface without protrusions. Sporocysts derived from the small intestine of the sparrowhawks measured 11.88 micromx8.34 microm. Polymerase chain reaction amplification and sequencing of the first internal transcribed spacer region (ITS-1), the 18S rRNA and the 28S rRNA gene comprising the variable D2 and D3 domains further characterized them as two novel Sarcocystis species. S. calchasi displays a pairwise distance value of the ITS-1 region ranging between 0.165 and 0.195 with the Sarcocystis spp. from the wood pigeon and the sparrowhawk, respectively. A phylogenetic analysis further supported the existence of two new species.

Light and transmission electron microscopic studies on the encystation of Histomonas meleagridis.

The study deals with the pleomorphic zooflagellate Histomonas meleagridis, which was cultivated under different stress conditions to induce a possible encystation. In the present paper, the morphological changes were analyzed by light and electron microscopy. The determination of the proliferation under different adverse conditions led to conclusions on the tenacity of the flagellate. H. meleagridis parasitizes in the intestinal tract of galliform birds and may cause enormous losses in poultry farming. For the development of new therapy approaches, clarification of the transmission pathways will be helpful. Different clonal cultures of H. meleagridis established by micromanipulation and exposed to media lacking different ingredients, inappropriate temperatures, and/or distinct reagents were investigated. Lowering of temperature was proven to have adverse effects on the survival of H. meleagridis. The flagellate could not survive in a frozen medium, and survival in a temperature of 4 degrees C lasted no longer than 23 h. An addition of sodium chloride induced an increased proliferation; pH values between 2 and 8 set limits for the survival of the parasite in different ways. H. meleagridis was able to survive under high acidic conditions for only 1 h. The major amount of cells, which could be discovered in the controls, measured 8-12 microm appeared amoebic (stage 1) and were filled with enclosures of rice starch. A rounding of most cells was noted 4 h at 4 degrees C after incubation in minimal essential medium in the absence of rice starch and fetal calf serum. A higher osmolarity of the medium, which was initiated by the addition of sodium chloride or magnesium chloride, did not induce an encystation process. After addition of hypochlorite base and cultivating at pH values between 7 and 8, spherical stages without a flagellum were formed (stage 2) measuring about 8-12 microm in diameter. Their interior consisted of a central and a peripheral region when studied by transmission electron microscopy. This aspect was due to the location of the glycogen granules. The central zone was described as totally filled with the carbohydrates, which made totally invisible the other organelles. The solidity of the amorphous layer below the cell membrane seemed to hinder the invasion of the glycogen granules. The amorphous layer below the cell membrane made it apparently possible that the cell might survive under adverse conditions-at least for a short time. This special structure might enable H. meleagridis to proceed a fast transmission and to infect many birds in a rather short time, which was shown in the past by several studies. Double-membraned cells, which were guessed to be cyst-like structures of the parasite, were also detected (stage 3). The size of these cells, however, was much smaller than that of the amoebic stages or the above-described spherical forms of H. meleagridis. Furthermore, the small cells were characterized by other granula structures. These findings might be interpreted that the small stages are possibly long-term (true) cysts and that the spherical stages with the amorphous layer beneath the cell membrane might be short-term cysts. Both, however, should be able to survive situations outside of a body and thus might be transmitted from feces to another animal.

Molecular biological comparison of different Besnoitia species and stages from different countries.

Besnoitia besnoiti tissue cysts from a recent outbreak in cattle in Germany were characterized with respect to their internal transcribed spacer regions 1, 2, and 18S rDNA gene sequences. These results were compared with own sequences of an Israelian isolate of B. besnoiti and of Besnoitia jellisoni cystozoites stored for years in liquid nitrogen. Furthermore, material was studied that was obtained from white mice (Balb/C) that had been successfully infected by intraperitoneal infection of fresh cystozoites from the German outbreak. All results were then compared and discussed with respect to databank sequences of other Besnoitia species. Comprehensive phylogenetic studies of B. besnoiti isolates from Germany revealed almost identical sequence alignments when compared to previously sequenced B. besnoiti isolates from Israel and Spain. More importantly, phylogenetic analysis revealed two distant clusters of Besnoitia species: the first one includes Besnoitia akodoni, Besnoitia darlingi, and Besnoitia oryctofelisi, while the second cluster includes B. besnoiti, Besnoitia bennetti, Besnoitia tarandi, and the Besnoitia species of rodents (B. jellisoni). The also B. jellisoni named species of the GenBank (AF 076860) must be another one, since our strain derives directly from Frenkel. These findings give strong hints that B. besnoiti has a cycle between rodents and a predator and that cattle and other are only accidental hosts.

Experiments to produce cysts in cultures of Histomonas meleagridis--the agent of histomonosis in poultry.

Experiments were done with cultured trophozoite stages of different clonal strains (Histomonas meleagridis/Turkey/Austria/2922-C6/04 and H. meleagridis/Chicken/Hungary/5009-C2/05) of H. meleagridis in order to induce a cyst formation as it is known in other intestinal parasites. It was shown that the best multiplication of H. meleagridis occurred at 40 degrees C in a full medium 199, when fetal calf serum and rice starch had been added. Under these conditions, numerous amoebic stages (8-12 microm in diameter) without and a few with flagellum were seen showing regular reproduction rates. When the conditions of culture were experimentally changed-and thus became worse-by decreasing the temperature, by deprivation of the medium from fetal calf serum and/or rice starch, and by changing the osmolarity, the pH, or the MgCl(2) concentration, many of the amoebic stages (containing starch granules) were destroyed, and several had obtained a spherical shape. If the culture conditions became even worse, smaller spherical stages occurred, which had only diameters of 4-7 microm and which appeared more condensed. Both spherical stages did not contain starch granules. All the previously seen stages disappeared constantly. Since a similar decrease of the optimal living conditions also occurs when intestinal or cloacal feces are deposited outside from the bird's body, the results obtained here may underline the interpretation that some of the formerly amoebic stages are able to become large spherical stages and later small spherical stages. The large spherical stage would be some type of precysts while the smaller ones would represent true cysts.

Sarcocystis calchasi sp. nov. of the domestic pigeon (Columba livia f. domestica) and the Northern goshawk (Accipiter gentilis): light and electron microscopical characteristics.

A novel highly pathogenic Sarcocystis species has been shown to cycle between the Northern goshawk (Accipiter gentilis) as definitive host and the domestic pigeon (Columba livia f. domestica) as intermediate host. However, genetically based characteristics are only available from very few bird-infecting Sarcocystis species. We therefore further characterised morphological properties of this protozoan in both hosts. Using light and electron microscopy, oocysts and sporocysts as well as schizonts and sarcocysts were characterised and compared with available morphological features of previously reported Sarcocystis species of Northern goshawks, Columbidae and genetically closely related species of other avian hosts. Sporocysts shed from day 6 on after experimental infection by the Northern goshawk were of ovoid appearance (11.9 x 7.9 microm). Ultrastructurally, schizonts of all developmental stages were found in the liver, spleen and next to or in endothelial cells of various organs of domestic pigeons 7 to 12 days after experimental infection. The cyst wall surface of slender sarcocysts (1 to 2 mm in length and 20 to 50 microm in width) was smooth and lacked protrusions. Cystozoites were lancet-shaped and measured 7.5 x 1.5 microm in Giemsa stain smears. The morphological findings, when combined with data of experimental infection and genetic studies, convergently indicate that the recently discovered Sarcocystis species represents a new species. We therefore propose to name this parasite Sarcocystis calchasi species nova.

Ultrastructure, development, and host-parasite relationship of a new species of the genus Pleistophora--a microsporidian parasite of the marine fish Epinephelus chlorostignei.

The life cycle of a new microsporidian of the genus Pleistophora is described. This parasite infects the epithelial cells of the gut and the peritoneal cavity of the Red Sea fish, Epinephelus chlorostignei. All stages develop within a special structure, the sporophorocyst, which is covered by a thick dense wall. This wall grows along with the growth of the parasites inside. Meronts are uni- to binucleate, which divide and constantly give rise to sporonts. During transition to sporonts, the cell border of the meronts increases its thickness, temporarily featuring thick irregular projections. Eventually, a uniform thick sporont wall is formed; then, the sporont cells detach themselves from the wall (future wall of the sporophorous vesicle, SPV) and start a series of divisions to produce sporoblasts. The SPV wall is compact, has no pores, and consists of two layers. Mature spores measure about 2.0 x 1.8 microm. They possess a polar filament with 20-28 coils, a posterior vacuole, and a polaroplast made up of an outer part of dense and closely spaced lamellae encircling an inner part of widely spaced lamellae. All morphological and ultrastructural features indicate that the described microsporidian parasite belongs to the genus Pleistophora.

Life cycle of Sarcocystis camelicanis infecting the camel (Camelus dromedarius) and the dog (Canis familiaris), light and electron microscopic study.

In the present study, the heteroxeneous life cycle of Sarcocystis sp. infecting camels were studied. A total of 180 slaughtered camels collected from different localities in Egypt were investigated for sarcocysts. Only 116 animals were found to be infected (the infection rate was 64%). Muscle samples of esophagus, diaphragm, tongue, skeletal, and heart muscles were examined. Exclusively, microscopic sarcocysts were detected in all examined organs. The infection rates of the esophagus, diaphragm, tongue, skeletal, and heart muscles were 60%, 50%, 40%, 40%, and 10%, respectively. By means of transmission electron microscopy, details of the ultrastructure of the sarcocysts were studied. The specific architecture and ornaments of the cyst wall, its protrusions, and the cyst interior were recorded. Unique features of protrusions of the primary cyst wall, the knob-like structures, arise around each protrusion. Experimental infection of carnivores by feeding heavily infected camel muscles revealed that the dog, Canis familiaris, is the only final host of the present Sarcocystis species. Gamogony, sporogonic stages, and characteristics of sporulated oocysts were also investigated.