Ultrastructural study on the development of Babesia equi (Coccidia: Piroplasmia) in the salivary glands of its vector ticks

The formation of Babesia equi sporozoites in the salivary glands of three tick species (Hyalomma anatolicum anatolicum, H. a. excavatum, Rhipicephalus turanicus) was studied by electron microscopy. The development was identical in all three vectors. On the 8th day post repletionem kinetes of B. equi had invaded alveoli of the nymphal salivary glands and were transformed to sporonts bounded by a single membrane. The sporonts were polymorphous bodies each with a highly lobed nucleus and numerous mitochondria. These stages persisted during ecdysis of the tick nymph to the adult stage. After attachment of these newly molted adults to a new host the formation of sporozoites was completed within five days. The sporonts occupied most of the infected alveolus and were extensively divided into cytoplasmic portions of various size. On the 4th day after attachment of the tick, sporozoite-anlagen, into each of which a nucleus and a mitochondrion were incorporated, appeared at the periphery of the sporonts. An apical complex with a polar ring, rhoptries, and micronemes was formed at the tip of each protruding anlage. Finally thousands of pyriform sporozoites (3.0 X 1.2 microns) filled the hypertrophied alveolus. This development is similar to sporogony in the genus Theileria.     

Ultrastructural Studies on the Trypanosomatid Phytomonas serpens in the Salivary Glands of a Phytophagous Hemipteran

Electron microscopy of salivary glands of the phytophagous hemipteran Phihia picta infected with Phytomonas serpens revealed the presence of flagellates in the gland periphery beneath the gland envelope, in the gland central lumen, between gland cells in the intercellular space and inside the gland cells. In the latter case, flagellates were found in the cytoplasm whether or not it was surrounded by a vacuolar membrane. Flagellates were always of the promastigote type, sometimes displaying a large twisted body. Morphological peculiarities of flagellates in different gland locations are recorded.     

Microbial Population Analysis of the Salivary Glands of Ticks; A Possible Strategy for the Surveillance of Bacterial Pathogens

Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.     

Na+-Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na⁺-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.     

Expression of Babesia equi merozoite antigen-2 by recombinant baculovirus and its use in the ELISA

In this study, the gene encoding Babesia equi merozoite antigen-2 was inserted into a baculovirus transfer vector, and a recombinant virus expressing B. equi merozoite antigen-2 was isolated. Two B. equi merozoite antigen-2-related recombinant baculovirus-expressed peptides of 25 and 30 kDa were detected with a murine anti serum against B. equi merozoite antigen-2; these corresponded to the native B. equi merozoite antigen-2 and were secreted into the supernatants of insect cell cultures. Recombinant B. equi merozoite antigen-2 was not effected by tunicamycin treatment, indicating that B. equi merozoite antigen-2 was not glycosylated. The potential of recombinant B. equi merozoite antigen-2 for use in the ELISA was evaluated by measuring the antibody response to B. equi merozoite antigen-2 in horses experimentally infected with B. equi.     

Ultrastructure of a Coccidium (Apicomplexa: Sporozoea: Coccidia) in Priapulus caudatus (Priapulida)1

Stages in the life cycle of a coccidium are described from the intestine of Priapulus caudatus Lamarck, 1816. Meronts, merozoites, microgamonts, microgametes, and walled and unwalled macrogametes were seen in intestinal cells. Meronts were about 8 μm long and 3–7 μm wide and produced up to seven merozoites. Free merozoites were about 9 μm long and 4 μm wide and contained about 43 subpellicular microtubules that terminated in the outer polar ring. Microgamonts were up to 23 μm long and 7 μm wide and usually were delimited by a single membrane. Microgametes were about 5 μm long, exclusive of the two flagella, about 2 μm wide, and contained a nucleus that was not uniformly dense. Macrogametes, about 6 μm in diameter, had a nucleus largely without dense chromatin. The oocyst wall formed around intracellular macrogametes to a thickness of 0.2–0.5 μm as thin, osmiophilic elements that became arranged in reticular and tubular layers. Wall-forming bodies were not seen, but fine filaments may participate in wall formation, as these were found between the outer membrane of the pellicle and the nearest wall elements. Microgametes and walled macrogametes were delivered to the lumen of the host intestine during apocrine secretion or excretion by the intestinal cells. Fertilization may occur in the intestinal lumen. Unsporulated ovoid oocysts, 18–27 μm long and 10–14 μm wide, with a 3 μm micropyle and a wall 0.6–0.7 μm thick, were passed from the host.     

Autophagy and Apoptosis Coordinate Physiological Cell Death in Larval Salivary Glands of Apis mellifera (Hymenoptera: Apidae)

Larval salivary glands of bees provide a good model for the study of hormone-induced programmed cell death in Hymenoptera because they have a well-defined secretory cycle with a peak of secretory activity phase, prior to cocoon spinning, and a degenerative phase, after the cocoon spinning. Our findings demonstrate that there is a relationship between apoptosis and autophagy during physiological cell death in these larval salivary glands, that adds evidence to the hypothesis of overlap in the regulation pathways of both types of programmed cell death. Features of authophagy include cytoplasm vacuolation, acid phosphatase activity, presence of autophagic vacuoles and multi-lamellar structures, as well as a delay in the collapse of many nuclei. Features of apoptosis include bleb formation in the cytoplasm and nuclei, with release of parts of the cytoplasm into the lumen, chromatin compaction, and DNA and nucleolar fragmentation. We propose a model for programmed cell death in larval salivary glands of Apis mellifera where autophagy and apoptosis function cooperatively for a more efficient degeneration of the gland secretory cells.

Addendum to:

Programmed Cell Death in the Larval Salivary Glands of Apis mellifera (Hymenoptera, Apidae)

E.C.M. Silva-Zacarin, G.A. Tomaino, M.R. Brochetto-Braga, S.R. Taboga, R.L.M. Silva de Moraes

J Biosci 2007; 32:309-28     

Development of a Model for the Role of Mitogens Initiating Repair Hyperplasia in Big Salivary Glands

It was shown in repeated experiments on 132 rats that proliferation in the corneal epithelium was nonspecifically enhanced after sialotomy. This effect appears to be due to the distant action of factors stimulating cell proliferation. The described phenomenon can be used for identification of mitogens formed during regeneration of the salivary glands.     

斯氏按蚊生长发育中唾液腺配子体激活因子的变化

为探讨斯氏按蚊生长发育过程中唾液腺抽提物配子体激活因子的消长,应用体外雄配子体出丝观察方法比较羽化后未吸血及吸血组雌性斯氏按蚊唾液腺抽提物中配子体激活因子对柏氏疟原虫雄配子体出丝诱导活性的动态变化。羽化后未吸血组按蚊唾液腺配子体激活因子活性与按蚊生长、发育呈同步变化。羽化后当日至羽化后第6 d,吸血组按蚊唾液腺抽提物的GAF活性变化与未吸血组相似,吸血后该组GAF活性下降,羽化后14 d恢复到吸血前水平。吸血后斯氏按蚊唾液腺配子体激活因子活性的降低与蚊卵发育可能相关。     

Seroprevalence of Babesia caballi and Theileria equi in the Swiss horse population

In Switzerland, the prevalence and incidence of equine piroplasma parasite (EPP) infections are unknown. In order to obtain a first insight into the prevalence, a representative sample of 689 sera of horses from Switzerland was serologically tested for the presence of antibodies directed against T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). A total of 50 (7.3%) horses were seropositive for EPP: overall, the seroprevalence of T. equi was significantly higher than that of B. caballi (p = 0.002). The seropositivities in indigenous horses (animals bred and raised in Switzerland) and in imported horses were 4.8% (11/230) and 8.5% (39/459), respectively. Unlike in indigenous horses, where no significant difference in seroprevalences could be observed between the two parasite species, the seroprevalence of T. equi was significantly higher (p < 0.001) than that of B. caballi in imported horses. Horses imported from France, Spain and Portugal exhibited a significantly higher seroprevalence, and horses imported from Germany a significantly lower seroprevalence of EPP compared to indigenous horses. There were no associations between sex, age, weight loss, surgery or blood transfusions with T. equi and B. caballi seroprevalences.The overall seroprevalence of 7.3% clearly shows that infection with EPP is a threat to the health of the horses in Switzerland. With the presumed expansion of permissive tick vectors, EPP infections will potentially increase in importance in the future. Therefore, continuous monitoring is mandatory.     

An Inhibitor of Thrombin-Stimulated Blood Platelet Aggregation from the Salivary Glands of the Hard Tick Amblyomma variegatum (Acari: Ixodidae)

The tropical bont tick, Amblyomma variegatum can cause intense skin irritation and inflammation and bites that often develop into septic wounds or abscess in their host. Crude salivary gland extract (SGE) of partially engorged A. variegatum females as well as SGE protein fractions purified by three-step reverse phase HPLC procedure were tested for their anti-aggregatory effect on isolated human blood platelets stimulated with thrombin and compared with the effect of recombinant hirudin. At concentrations 10⁻³ and 5 × 10⁻³ μg protein/ml the following rank order of antiplatelet activity was detected: AV 16/3 (inhibitor purified from AV-III, third purification) > SGE > AV-II (fraction from first purification) > AV-III (fraction from first purification) > hirudin. The effect of all fractions tested was dose-dependent. For fraction AV 16/3, the inhibitory effect was 49 and 61% for 10⁻³ and 5 × 10⁻³ μg protein/ml, respectively. The results suggest that protein fractions from A. variegatum SGE possess an antithrombin effect on human blood platelets with hirudin-like activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ticks (Acari: Ixodida) on wild carnivores in Brazil

The present study reports field data of ticks infesting wild carnivores captured from July 1998 to September 2004 in Brazil. Additional data were obtained from one tick collection and from previous published data of ticks on carnivores in Brazil. During field work, a total of 3437 ticks were collected from 89 Cerdocyon thous (crab-eating-fox), 58 Chrysocyon brachyurus (maned-wolf), 30 Puma concolor (puma), 26 Panthera onca (jaguar), 12 Procyon cancrivorus (crab-eating-raccoon), 4 Speothos venaticus (bush-dog), 6 Pseudalopex vetulus (hoary-fox), 6 Nasua nasua (coati), 6 Leopardus pardalis (ocelot), 2 Leopardus tigrinus (oncilla), 1 Leopardus wiedii (margay), 1 Herpailurus yagouaroundi (jaguarundi), 1 Oncifelis colocolo (pampas-cat), 1 Eira barbara (tayara), 1 Galictis vittata (grison), 1 Lontra longicaudis (neotropical-otter), and 1 Potus flavus (kinkajou). Data obtained from the Acari Collection IBSP included a total of 381 tick specimens collected on 13 C. thous, 8 C. brachyurus, 3 P. concolor, 10 P. onca, 3 P. cancrivorus, 4 N. nasua, 1 L. pardalis, 1 L. wiedii, 4 H. yagouaroundi, 1 Galictis cuja (lesser grison), and 1 L. longicaudis. The only tick infested-carnivore species previously reported in Brazil, for which we do not present any field data are Pseudalopex gymnocercus (pampas fox), Conepatus chinga (Molina’s hog-nosed skunk), and Conepatus semistriatus (striped hog-nosed skunk). We report the first tick records in Brazil on two Felidae species (O. colocolo, H. yagouaroundi), two Canidae species (P. vetulus, S. venaticus), one Procyonidae species (P. flavus) and one Mustelidae (E. barbara). Tick infestation remains unreported for 5 of the 26 Carnivora species native in Brazil: Oncifelis geoffroyi (Geoffroy’s cat), Atelocynus microtis (short-eared dog), Pteronura brasiliensis (giant otter), Mustela africana (Amazon weasel), and Bassaricyon gabbii (olingo). Our field data comprise 16 tick species represented by the genera Amblyomma (12 species), Ixodes (1 species), Dermacentor (1 species), Rhipicephalus (1 species), and Boophilus (1 species). Additional 5 tick species (3 Amblyomma species and 1 species from each of the genera Ixodes and Ornithodoros) were reported in the literature. The most common ticks on Carnivora hosts were Amblyomma ovale (found on 14 host species), Amblyomma cajennense (10 species), Amblyomma aureolatum (10 species), Amblyomma tigrinum (7 species), Amblyomma parvum (7 species), and Boophilus microplus (7 species).This revised version was published online in September 2005 with corrected page numbers.     

Coccidia in the mammary glands of shrews (Order: Insectivora)

Three of 6 female long-clawed shrews, Sorex unguiculatus Dobson, 1890, collected on the island of Hokkaido, Japan, were found to have unsporulated oocysts and sexual stages (both macro- and microgamonts) in varying stages of development of an unidentified coccidium in both lactating and nonlactating mammary glands. Gamonts developed in the alveoli of the mammary glands, and oocysts were found in the lactiferous ducts and in pools of milk. Mature macrogamonts were 11.9 x 15.2 microm (10-14 x 14-20 microm), whereas completely developed microgamonts with many gametes were 14.8 x 16.8 microm (10-18 x 13-20 microm). Oocysts in tissue sections were 19.5 x 13.8 microm and had a smooth outer wall that was <1 microm thick. Little histopathology was associated with the infections. Infected cells were enlarged and appeared cloudy, and in some areas there was leucocytic infiltration by macrophages, small and large lymphocytes, neutrophils, and eosinophils. No basophil was seen. We also found sections of a nematode, probably a Mammanidula sp., in sections of an active mammary gland in 1 of the shrews not infected with the coccidium.     

An Ultrastructural Study of Phytomonas davidi Lafont (Trypanosomatidae)1

Phytomonas davidi (Trypanosomatidae) possesses typical trypanosomatid organelles: subpellicular microtubules, kinetoplast-mitochondrial complex, K-DNA, and four subflagellar pocket microtubules. A greater concentration of subpellicular microtubules was observed in the latex forms than in those found in the salivary glands of its insect vector. Only in the latex flagellates (the stage with postnuclear torsion) were subpellicular microtubules interconnected by crossbridges observed. Morphology and development of mitochondrial aristae varied according to the source of the flagellates. Organisms taken from culture medium had extensively developed plate-like cristae; sparse tubular cristae were observed in the latex forms; and highly developed tubular cristae were seen in flagellates from the lumen of the vector's salivary glands, though organisms in the salivary gland channels had few or none.