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.

Molecular biological features of strains of Histomonas meleagridis.

Berlin strains of Histomonas meleagridis were subcultivated to produce cyst-like stages. These strains were studied for their ITS 1 and 18S rRNA properties and compared with sequences in data banks of other H. meleagridis strains, Dientamoeba fragilis, and some species of the genus Trichomonas and relatives. The Berlin isolates that had previously been shown to be able to develop cyst-like structures (Munsch et al. 2008) represent a significant cluster among the published data of other Histomonas meleagridis isolates and thus the formation of cysts might be a common feature that would open further possibilities of transmission.

Genebank accession numbers of sequences of Culicoides species vectors of bluetongue virus in Germany.

The paper offers the genebank accession numbers of Culicoides obsoletus, Culicoides scoticus and Culicoides pulicaris sequences (ITS 1, ITS 2, 18S rRNA) that had been shown to be vectors of the bluetongue virus serotype 8, which was introduced in 2006 into Germany and spread until 2009 all over Central Europe, including parts of England. The numbers are FN 263292 until FN 263323.

Transmission of feline calicivirus via the cat flea (Ctenocephalides felis).

In this study, a possible role of the cat flea (Ctenocephalides felis) in transmitting feline calicivirus (FCV) was examined. Fleas were fed via artificial membranes with FCV-spiked bovine blood, free of anti-FCV antibodies. Flea feces were collected daily for 10 days and incubated at room temperature. Infectivity of the feces was tested in vitro using Crandell-Reese Feline Kidney (CRFK) cells. FCV remained infectious for 8 days. These flea feces were also used to oronasally inoculate four specific pathogen-free (SPF) kittens. All kittens were successfully infected as demonstrated by virus isolation from pharyngeal swabs and seroconversion. Two of the cats showed, in addition, clinical signs. Besides the infection of cats with flea feces containing FCV, four SPF kittens were exposed to fleas that were fed with FCV-spiked bovine blood. One of the kittens was successfully infected via this route as demonstrated by virus isolation from pharyngeal swabs and virus isolation. The results of this study show that fleas can spread infectious virus through their feces or by stitch and must be considered a source of infection for uninfected cats.

Determining a diagnostic dose for imidacloprid susceptibility testing of field-collected isolates of cat fleas (Siphonaptera: Pulicidae).

The susceptibility of four laboratory strains of cat fleas, Ctenocephalides felis (Bouche), to imidacloprid was determined by three different laboratories, by using a standardized bioassay protocol. The probit lines generated by the different laboratories were very similar, with LC50 values ranging from 0.32 to 0.81 ppm. Based on these data, a diagnostic dose (DD) of 3 ppm imidacloprid in larval rearing media was provisionally identified for detecting shifts in tolerance, possibly as a consequence of incipient imidacloprid resistance. None of the larvae from the susceptible laboratory strains survived the DD. Eighteen field-collected isolates were evaluated for their susceptibility to imidacloprid and to validate a DD of 3 ppm. Probit lines from 18 field-collected isolates were very similar, with LC50 values ranging from 0.14 to 1.52 ppm. When exposed to the DD, between 3 and 10% of the exposed larvae emerged as adults from only three of the 18 isolates. All other field isolates gave 100% mortality at the DD. Under the criteria established (>5% survivorship at 3 ppm), two isolates would be established on mammalian hosts and more extensive tests conducted to exclude or confirm the presence of resistance. The DD of 3 ppm is robust enough to eliminate most of the susceptible isolates collected until today, yet low enough to identify possible isolates for further testing.

Efficacy of a combination of imidacloprid and moxidectin against parasites of reptiles and rodents: case reports.

A new compound containing imidacloprid 10% (w/v) and moxidectin 2.5% (w/v) (Advantage multi, Advocate) was applied as a spot-on treatment to mice experimentally infected with Trichuris muris. Case reports of reptiles found positive for nematode and mite infections following parasitological examination and treated with this compound are also discussed. The results demonstrated that the registered, recommended 2.5% moxidectin concentration for use in dogs was sufficient to eliminate nematodes and mites in reptiles. Infections with nematodes were successfully treated with a single application. Mite infestations in reptiles were eliminated using a treatment repeated on 3 consecutive days.

Experimental quantification of the feline leukaemia virus in the cat flea (Ctenocephalides felis) and its faeces.

Cat fleas (Ctenocephalides felis) were fed via artificial membranes and infected with the feline leukaemia virus (FeLV) from cell cultures. After removing the fleas from the blood source, the quantity of virus in the flea and its faeces was measured over a defined period of time. The virus was detectable in the fleas for up to 30 h at room temperature and up to 115 h at 4 degrees C. In the faeces, the amount of virus decreased much more slowly--after 2 weeks half of the initial amount of virus could still be detected. Thus the faeces might be a source of further infections, e.g. for the flea larvae or the cat itself.

Field trial of the efficacy of a combination of imidacloprid and permethrin against Tunga penetrans (sand flea, jigger flea) in dogs in Brazil.

In a field trial in Brazil 17 dogs penetrated by females of the jigger flea, Tunga penetrans, were topically treated with a combination of 10% imidacloprid and 50% permethrin (Advantix), while 17 dogs remained untreated. The follow-up controls on days 7, 14, 21 and 28 post-treatment clearly showed that, beginning from day 7, the flea load in treated dogs decreased, so that most of the dogs became free of tungiasis lesions, while in the untreated group the flea load remained high. Since the dogs distribute the flea eggs throughout the village, leading to a high incidence of tungiasis in humans, treatment of dogs probably also decreases the number of cases of tungiasis in the latter.

Effects of a combinations of emodepside and praziquantel on parasites of reptiles and rodents.

A new combination of two anthelmintic compounds containing emodepside and praziquantel (Profender, Bayer AG, Levekusen, Germany) was tested in pet rodents and reptiles. Topical application of the two compounds led to the quick disappearance of nematodes and cestodes from a broad spectrum of hosts including mice, jirds, snakes, anole lizards, turtles, monitor lizards, etc. In reptiles the dosage had to be increased, since the thick outer layer of the epidermis hinders the penetration of the compounds. In animals with an extremely thick epidermis (e.g. monitor lizards, leguans) the new product was applied under the armpits.

Ascorbate is the natural substrate for plant peroxidases.

Ascorbate-dependent detoxification of hydrogen peroxide by guaiacol-type peroxidases is increased considerably in the presence of 3,4-dihydroxyphenolic compounds, suggesting that ascorbate is the natural substrate for many types of peroxidase in situ and not just the ascorbate-specific peroxidases. The ascorbate-dependent destruction of hydrogen peroxide in the more acidic cellular compartments such as the vacuole may be an important function of such non-specific peroxidases. The stress-induced production of phenolic compounds would render the guaiacol peroxidases in other less acidic-cellular sites effective as ascorbate-dependent H2O2-detoxifying enzymes.

Characterization of cDNA clones encoding a major microneme antigen of Sarcocystis muris (Apicomplexa) cyst merozoites.

Two monoclonal antibodies directed against a microneme antigen of Sarcocystis muris cyst merozoites (16/17 kDa band doublet) were used to isolate cDNA clones from a lambda ZAP expression library. Restriction analysis revealed that the inserts were highly similar, with sizes ranging between 1.8 and 2.3 kb. In addition, a full-length cDNA insert of 2.6 kb was obtained by hybridization screening. On Northern blots, a single mRNA species of 2.7 kb was detected by a cDNA-derived probe. Southern blot hybridization suggests that the gene is present as a single copy. The nucleotide sequence of the full-length clone contains a single reading frame with a coding capacity of 26.5 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide followed by a hydrophilic domain of unknown function, and the mature protein sequence. After purifying the 16/17 kDa antigen from cyst merozoites, a partial N-terminal amino acid sequence was obtained. Thus, the identity of the cDNA sequence was confirmed. The deduced sequence of the mature protein is predominantly hydrophilic and rich in cysteine (8.7%). Database searching suggested weak homologies of the hypothetical polypeptide to plasma kallikrein, tenascin and blood coagulation factors.

Cloning and expression in Escherichia coli of cDNAs encoding a 31-kilodalton surface antigen of Sarcocystis muris.

Using polyadenylated RNA isolated from Sarcocystis muris cyst merozoites, we have constructed a cDNA library in the expression vector lambda ZAP. Immunoscreening with monoclonal and polyclonal antibodies directed against a 31-kDa surface antigen of S. muris [1] yielded a number of clones with insert sizes ranging between 1.1 kb and 1.3 kb. An additional clone with an insert length of 1.55 kb was isolated by screening with a labeled DNA probe derived from one of the cDNA clones. The cDNA sequence was found to contain an open reading frame specifying a polypeptide of 280 amino acids with a predicted size of 29.7 kDa. The deduced amino acid sequence is rich in serine and threonine (22%) and harbors a hypothetical N-terminal signal peptide sequence as well as a C-terminal glycosyl phosphatidylinositol anchor attachment site. The predicted amino acid sequence has been confirmed by peptide sequencing and an analysis of the overall amino acid composition of the 31-kDa protein. A recombinant protein was obtained which was recognized by the polyclonal antibodies directed against the 31-kDa antigen. Antiserum raised against the purified fusion protein specifically reacted with a 31-kDa protein from S. muris cystozoites. Southern blot analysis indicated that the corresponding gene exists as a single copy within the S. muris genome.

New results on the effect of praziquantel in experimental cysticercosis.

14C-praziquantel penetrates the cyst wall of Cysticercus fasciolaris and kills the cysticercus within the cyst, although the uptake of praziquantel by the encysted larva was slower than by an isolated one. This fact is in good agreement with earlier in vitro chemotherapeutic studies. Scanning and transmission electron microscopic studies have shown that praziquantel causes a marked destruction of the tegument along the whole pseudostrobila and the scolex of C. fasciolaris. The type of tegumental damage is identical to that produced in adult tapeworms and trematodes.

Electron microscopic studies on the development of kinetes of Theileria parva Theiler, 1904 in the gut of the vector ticks Rhipicephalus appendiculatus Neumann, 1901.

The development of motile stages, called kinetes, from a stationary stage (regarded as zygote) has been followed in Theileria parva by means of electron miscroscopy. This process started after moult of the tick nymphs which had sucked on highly infected calves, i.e. about 20 days after repletion (a.r.) of the ticks. The transformation took place by formation of a growing protrusion (= anlage) into an inner, enlarging vacuole. During this process the limiting membrane of the enlarging vacuole serves as the outer membrane of the developing motile stage, whereas the two inner ones as well as the subpellicular microtubules are newly formed. This transformation proceeds rapidly, so that on the 25th day a.r. most of the kinetes have already left the gut cells and started penetration into the salivary gland cells. On the way to the salivary glands nuclear divisions occurred within the kinetes. The steps of the transformation described were compared to those in T. annulata and to ookinete formation in haemosporidia.

Electron microscopical studies on the development of Babesia canis (Sporozoa) in the salivary glands of the vector tick Dermacentor reticulatus.

The formation of sporozoites of Babesia canis was studied by light- and electron microscopy in the salivary gland cells of adult female ticks from the 2nd day after attachment until 1 day after detachment. It was found that this process was initiated by the binary division of kinetes that had already entered or entered during the period examined. During division the kinetes (15 X 2.5 microns) lost their typical organelles, reduced their three-layered pellicle to a single membrane and became spherical. After nuclear division and a further time-lag cell division occurred, giving rise to two cells in which this process was repeated. After numerous binary divisions the parasites acquired more and more closely the shape of the later infectious, pyriform sporozoite. These sporozoites measured about 2.5 X 1.5 microns and also had a three-layered pellicle, with rhoptries and a few micronemes, but never contained "spherical bodies". The formation process needed about 2--3 days so that the transmission to the dog could be carried out while the tick engorges and this is probably the stimulation for the development. Finally the cytological features of this sporozoite formation were compared to those in the Theileria species studied by our group.

Electron microscopic study on the development of Babesia ovis (Piroplasmia) in the salivary glands of the vector tick Rhipicephalus bursa.

The formation of Babesia ovis sporozoites in salivary gland cells of the vector tick Rhipicephalus bursa was studied by electron microscopy. The kinetics of B. ovis were found lying intracellularly on the second day after infestation (a.i.) of the ticks. The parasites enlarged rapidly losing all features of the motile form. Invaginations of the cell membrane initiated a fragmentation of this developmental stage. On the third day a.i the parasite (measuring 40 x 25 microns) was divided into numerous single membrane-bounded cytomeres, each provided with at least one lobed nucleus. On the fourth day a.i. sporozoite differentiation started at the periphery of the cytomeres, indicated by the appearance of several pellicle-bounded, exogenous protrusions into each of which a small portion of the nucleus was incorporated. Since the cytomeres lay very close together this differentiation occurred more by segmentation than by budding. Rhoptries and the so-called spherical body appeared in this developmental phase. Finally, the isolated, immature sporozoites lay in a granular matrix which contained remnants of the host cell cytoplasm. On the fifth day a.i. the sporozoites were fully developed, typically pear-shaped (2.8 x 1.2 microns) and provided with all characteristic structures of the invasive form.--This reproduction was compared to similar processes in other species of the Piroplasmia and the Haemosporina.

Development of a larval bioassay for susceptibility of cat fleas (Siphonaptera: Pulicidae) to imidacloprid.

Strategies for controlling cat fleas, Ctenocephalidesfelisfelis (Bouché), have undergone dramatic changes in the past 5 yr. With the advent of on-animal treatments with residual activity the potential for the development of insecticide resistance increases. A larval bioassay was developed to determine the baseline susceptibility of field-collected strains of cat fleas to imidacloprid. All four laboratory strains tested showed a similar level of susceptibility to imidacloprid. Advantages of this bioassay are that smaller numbers of fleas are required because flea eggs are collected for the test. Insect growth regulators and other novel insecticides can also be evaluated. Using a discriminating dose, the detection of reduced susceptibility in field strains can be determined with as few as 40 eggs.

Treatment of fish parasites: 6. Effects of sym. triazinone (toltrazuril) on developmental stages of Glugea anomala, Moniez, 1887 (microsporidia): A light and electron microscopic study.

A symmetric triazinone (toltrazuril) was tested in vivo against Glugea anomala parasitizing the connective tissue of sticklebacks (Gasterosteus aculeatus). Naturally infected sticklebacks were incubated in water containing 0, 5, 10 or 20 µg toltrazuril/ml or in pure solvent (4 ml/1000 ml water). In addition, treatment was done by intermittent therapy (6 × 5 or 20 µg/ml for 4 or 1 h, respectively, in two days intervals). After single treatment the drug caused significant damages on uni- or multinucleate meronts, sporogonial plasmodia, sporoblasts and immature spores. The damages mainly consisted in a decrease of the number of ribosomes, a reduction of the multinucleate meronts, a disturbance in the formation of the sporophorous vesicles, in general a lysis of the karyoplasm and malformations of the polaroplast. The extent of damages was correlated with the dose of the drug administered. After intermittent therapy the damages described above were intensified; the multinucleate meronts and the sporogonial plasmodia then disappeared. However, even by intermittent treatment the mature spores were not affected. It is suggested that chemotherapy of Glugea - infected fishes may be accomplished by bathing the fishes in separate, aerated containers by means of interval treatment (six times with 5 or 20 µg toltrazuril/ml for 4 h, respectively 1 h in two days intervals). The treatment will decrease considerably the output of spores. However, since the mature spores are not affected, a repetition of the interval treatment within several months is recommended. Fish with extended skin lesions, caused by net catching, or infections by fungi should be carefully observed during the treatment, because these factors decrease their drug tolerance.

Treatment of fish parasites: 5. The effects of sym. triazinone (Toltrazuril) on fish parasitic ciliophora (Ichthyophthirius multifiliis FOUQUET, 1876, Apiosoma amoebea GRENFELL, 1884, Trichodina sp. EHRENBERG, 1831).

For chemotherapy of fish parasitized by different Ciliophora (Protozoa) toltrazuril was tested in vivo and in vitro against skin and gill parasitizing species (e.g. Ichthyophthirius multifiliis, Apiosoma amoebea and Trichodina sp.). For in vitro treatment naturally infected fish were incubated at 20 °C for 0.3,1,2,4.5 h in water containing 0,1,5,10,20 or 50 µg toltrazuril/ml. Lethal damages already occurred in about one third of the trophozoites of I. multifiliis after incubation in 5 µg/ml for 4.5 h, and in more than two thirds of the trophozoites after incubation in 10 µ/ml for 2 h. A few trophozoites, however, were still able to leave their hosts, to encyst and to produce theronts. However, when treatment was done by intermittent therapy (1×10 µg/ml for 2 h, first day; 1 × 20 µg/ml for 1 h, second day; 1 × 20 µg/ml for 1 h, third day) all the trophozoites were lethally damaged. The damages consisted in the destruction of the outer cell membranes, of the cilia, and of the mitochondria, as well as in the complete abolishment of the ribosomes and in the enlargement of the nuclear space. After in vitro treatment (10 µg/ml, 2 h) all the trophozoites were lethally damaged. In contrast to the trophozoites, the free-swimming theronts of I. multifiliis were not affected by the drug. In vivo treatment starting with 20 µg/ml for 1 h led to severe damages in A. amoebea, which were intensified after treatment with 50 µg/ml for 0.3 h. The specimens were heavily contracted, and the oral cilia were redrawn. Furthermore, the pellicular pores seen in the surface of controls were not detectable. Despite the affections caused by the treatment the parasites did not drop off their hosts. In vivo treatment against Trichodina sp. led to a reduced motility in these parasites starting with 10 µg/ml for 2 h. Incubation with 20 µg/ml for 1 h caused a complete stop of motion in most of the specimens. The highest dose (50 µg/ml; 0.3 h) only caused a dropping off in about one third of the specimens from their hosts. Treated specimens of Trichodina sp. had a more flattened appearance compared to untreated controls, and the epistomial disc was drastically enlarged. From these experiments it is suggested that treatment against the trophozoites of I. multifiliis should be done by intermittent therapy according to the following scheme: 1st day: 10 µg/ml, 2 h, 2nd day: 20 µg/ml, 1 h; 3rd day: 20 µg/ml, 1 h. In the cases of Apiosoma spp. and Trichodina spp. infected fish incubating with 50 µg/ml for only 20 minutes is recommended.