Ultrastructure of sporogony in Babesia equi in salivary glands of adult female Boophilus microplus ticks

The development of Babesia equi was studied in the salivary glands of adult female ticks, Boophilus microplus, using a transmission electron microscope (TEM). Engorged nymphs were obtained from splenectomized foals experimentally infected with B. equi and fed in the adult phase for 5 days on rabbits. Sporogony in B. equi involves the development of sporoblasts and sporozoites, which form from finger-like projections on the surface and through radial budding. Mature sporozoites (2.0 × 1.1 μm), typically pyriform, showed a polar ring, rhoptries, micronemes, nuclei, and mitochondria, and a high concentration of free ribosomes were observed from the 2nd day of the ticks, feeding on the rabbits. In general, sporogony of B. equi in the salivary glands of B. microplus showed similarities to the development of this parasite in species of Hyalomma, although with some significant differences in the sporozoite's dimensions. The results of this study indicate that B. equi is capable of multiplying in the salivary glands of adult female B. microplus, forming sporozoites with specialized organelles characteristic of the invasive form, and suggest that B. microplus can act as a natural vector of B. equi in endemic areas where there is no other probable source of infection or where it is the only tick species present on horses. Received: 5 February 1997 / Accepted: 14 May 1997     

New findings in the development ofBabesia (Theileria) equi (Laveran, 1901) in the salivary glands of the vector ticks,Hyalomma species

The development of the piroplasmBabesia equi was studied by light microscopy in the salivary glands of three differentHyalomma species during and after the engorgement of nymphs on experimentally infected horses and after adults had fed on a vertebrate host following ecdysis. The stock ofB. equi used was isolated from a horse imported from Turkmenistan (CIS) in 1991. The findings, being identical in all threeHyalomma species, differ with regard to the chronological order of the development stages in several respects from the results of previous studies based upon light or electron microscopy. A first sporogony phase ofB. equi was found to develop in the salivary glands of the engorged nymphs before the ticks moulted to adults. Beginning at day 6 postinfestation (p. infest.) of the nymphs, spindle-shaped sporozoites appeared to be formed by both rapid sequential fission of a multinucleated complex and a process of radial budding from multiple fission bodies. Sporozoites isolated from the salivary glands of the engorged nymphs proved to be infectious when they were injected into a susceptible horse. After the nymphs had moulted, a second sporogony phase similar to the first one observed in the salivary glands of engorged nymphs could also be initiated in the salivary glands of adults when they were attached to a vertebrate host. Sporozoites produced in the salivary glands of adults were equally infectious for horses. Thus, two completely separate sporogony phases inB. equi seem to develop successively in the salivary glands ofHyalomma species during a transstadial transmission.     

Detection ofTheileria parva in the salivary glands ofRhipicephalus appendiculatus: evaluation of staining methods

A comparison of ten methods for staining tick salivary glands for detection ofTheileria parva infection from ticks fed on rabbits for various periods was undertaken. Staining with azure without hydrochloric acid hydrolysis was found to be the most reliable method for detection of the presporozoite stages (sporoblasts) ofT. parva in the salivary gland acini of unfedRhipicephalus appendiculatus and could be applied to field ticks. All the stains proved suitable for the detection and quantitation of sporozoites in ticks fed for 4 days on rabbits. The capacity of the stains to allow detection of early stages ofT. parva differed, but it became more reliable during tick feeding as sporoblasts developed and matured. Giemsa's stain and Feulgen's stain followed by superimposition of Giemsa's stain were superior to other stains for the detection and quantitation of immature salivary gland stages in feeding ticks.     

Persistently infected horses are reservoirs for intrastadial tick-borne transmission of the apicomplexan parasite Babesia equi

Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of ≥10⁴ organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.     

Ticks’ response to feeding on host immunized with glandular extracts of <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> females fed for 2, 4, and 6 days. I. Inactivity or early degeneration of salivary glands?

The present study histologically analyzed the salivary glands of Rhipicephalus sanguineus females fed for 2, 4, and 6 days in hosts which had been previously immunized with glandular extracts obtained from females from this same species in different periods of feeding, having as main objective verify the action of these extracts in the secretor cycle of these glands. For this, glandular extract of females fed for 2 days (SGE2), glandular extract of females fed for 4 days (SGE4), and glandular extract of females fed for 6 days (SGE6) extracts were obtained from salivary glands of R. sanguineus females fed for 2, 4, and 6 days respectively. Then, New Zealand White naive rabbits were inoculated either with extracts (test group = TG), or with a mixture of phosphate buffer and Freund's complete adjuvant (control group 2 = CG2). Each inoculated rabbit (TG and CG2) and non-inoculated (control group 1 = CG1) was posteriorly infested with 15 couples of fasting R. sanguineus from which the salivary glands had been collected from females fed for 2, 4, and 6 days. The results revealed that the resistance the hosts had acquired by the immunization with the extracts affected differently the secretory activity of the glandular cells. It was verified that the resistance to SGE2 and SGE4 extracts acted in the cells of acini II and III, being c1 and c5 from II and d from III inactivated due to the action of SGE2 and c1 and c4 from II and f from III inactivated by the action of SGE4. As for the resistance to SGE6 the effect was only on cells of acini II (c1, c3 e c4), which were also inactivated. In addition, the hosts’ resistance to SGE2–SGE6 extracts made the degenerative process earlier in comparison to CG1. On the other hand, the resistance to the extracts did not influence the characteristics of the degenerative process normally found in salivary glands. The assynchronism of the degenerative process was maintained—acini III were always the most affected and I the less affected. The structural cell alterations, such as cytoplasmic vacuolation, nuclear alterations and formation of apoptotic bodies which characterize the occurrence of atypical apoptosis were also maintained in the glands of individuals from TG making it clear that the immunization of the hosts with glandular extracts SGE2–SGE6 had influenced the glandular physiology of R. sanguineus, which is an important piece of information in the search for a way to control these ectoparasites.     

Cell Death in Salivary Glands of Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) Females at Semi-engorged Feeding Stage

The ultrastructure of the salivary glands of Rhipicephalus (Boophilus) microplus females is described during feeding. In beginning of feeding, individuals show acini I with many mitochondria and wide basal labyrinth in peripheral cells; glycoprotein granules only in b and c₃ cells (acini II); and epithelial interstitial cells with developed basal labyrinth between f cells (acini III). Semi-engorged females show cells in degeneration, with autophagic vacuoles, lysosomes, myelin figures, and irregular, condensed, and/or fragmented nuclei, in addition to apoptotic bodies. R. B. microplus points to apoptosis in these organs before the detachment from the host, in contrast to others tick species.     

DNA probes detectTheileria parva in the salivary glands ofRhipicephalus appendiculatus ticks

The ability ofTheileria parva-specific DNA probes to detectT. parva sporoblasts and sporozoites in samples prepared from the salivary glands of infectedRhipicephalus appendiculatus ticks was evaluated. The two DNA probes used, pgTpM-23 and lgTpM-58, were selected from a genomic library ofT. parva (Muguga) piroplasm DNA. In all, 25–200 adult ticks infected with each of 6 differentT. parva stocks were tested. One salivary gland from each tick was processed for DNA hybridization, whereas the other was stained and examined by light microscopy to determine the number of infected acini. The correlation for the detection of infected acini between the two methods was 90%–100% for both probes, except when the pgTpM-23 probe was hybridised to salivary glands from ticks infected with the Mariakani stock ofT. parva (84% correlation). The discrepancy lay within the range expected, based on the observation that in 12.5% of the ticks, only one salivary gland was infected. The probes did not hybridize to salivary glands from uninfected ticks.     

Plasmodium falciparum andP. berghei: detection of sporozoites and the circumsporozoite proteins in the saliva ofAnopheles stephensi mosquitoes

Sporozoites and free circumsporozoite (CS) protein were stained immunoenzymatically in 1-min saliva samples collected fromAnopheles stephensi mosquitoes infected with eitherPlasmodium berghei orP. falciparum. The number of sporozoites in 1-min saliva-streak samples significantly increased as the salivary gland index rose from 3+ to 4+. ForP. berghei-infected mosquitoes from which saliva had been collected before 30 days postfeed, the median sporozoite counts for 3+ and 4+ gland indexes were 4.5 and 116, respectively. ForP. falciparum-infected mosquitoes, the median counts obtained in two experiments were 4.5 and 14.5 (3+) and 97 and 107 (4+), respectively. The frequency of sporozoite detection in the saliva of mosquitoes containing <100 salivary-gland sporozoites was low (0.1), whereas that in the saliva of mosquitoes with >100 sporozoites was high (0.96). In highly infected 4+P. berghei-infected mosquitoes from which saliva had been collected after 30 days postinfection, both the volume of saliva collected and the median number of sporozoites recovered decreased significantly.     

Plasmodium berghei Sporozoites Acquire Virulence and Immunogenicity during Mosquito Hemocoel Transit

Malaria is a vector-borne disease caused by the single-cell eukaryote Plasmodium. The infectious parasite forms are sporozoites, which originate from midgut-associated oocysts, where they eventually egress and reach the mosquito hemocoel. Sporozoites actively colonize the salivary glands in order to be transmitted to the mammalian host. Whether residence in the salivary glands provides distinct and vital cues for the development of infectivity remains unsolved. In this study, we systematically compared the infectivity of Plasmodium berghei sporozoites isolated from the mosquito hemocoel and salivary glands. Hemocoel sporozoites display a lower proportion of gliding motility but develop into liver stages when added to cultured hepatoma cells or after intravenous injection into mice. Mice infected by hemocoel sporozoites had blood infections similar to those induced by sporozoites liberated from salivary glands. These infected mice display indistinguishable systemic inflammatory cytokine responses and develop experimental cerebral malaria. When used as metabolically active, live attenuated vaccine, hemocoel sporozoites elicit substantial protection against sporozoite challenge infections. Collectively, these findings show that salivary gland colonization does not influence parasite virulence in the mammalian host when sporozoites are administered intravenously. This conclusion has important implications for in vitro sporozoite production and manufacturing of whole-sporozoite vaccines.     

Improved disease resistance after Babesia divergens vaccination

The efficacy of a new inactivated vaccine against Babesia divergens was evaluated by means of inoculation tests. The infection was initiated by i.v. injection of blood containing 2 × 10⁹ living parasites into splenectomized and non-splenectomized calves. Clinical status and hematological parameters were determined. Serology examinations for antibodies against B. divergens were carried out by indirect fluorescent antibody test (IFAT). Non-vaccinated and splenectomized animals exhibited experimental infections. In vaccinated and splenectomized animals, clinical symptoms and prolonged incubation periods were observed. Received: 16 July 1997 / Accepted: 4 September 1997     

Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis

The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 × 10⁻³% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 × 10⁻³% T. equi and 1 × 10⁻¹% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture increased up to 1 × 10⁻⁶%, whereas LAMP detection limit increased to 1 × 10⁻¹⁰% for both parasites, whilst the PCR detection limit increased to 1 × 10⁻¹⁰% and 1 × 10⁻⁶% for T. equi and B. caballi, respectively. Furthermore, LAMP and PCR amplified the T. equi DNA extracted from the organs of an experimentally infected horse. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of equine piroplasmosis and together with PCR can also be used as supplementary methods during post-mortems.     

Protective Effects of Passively Transferred Merozoite-Specific Antibodies against Theileria equi in Horses with Severe Combined Immunodeficiency

Theileria equi immune plasma was infused into young horses (foals) with severe combined immunodeficiency. Although all foals became infected following intravenous challenge with homologous T. equi merozoite stabilate, delayed time to peak parasitemia occurred. Protective effects were associated with a predominance of passively transferred merozoite-specific IgG3.     

Studies on the viability of sporozoites of Eimeria tenella

Summary Sporozoites, obtained from oocyst cultures stored at 4° C for more than 65 days were markedly less viable than those hatched from new oocyst cultures. There appeared to be no quantitative differences in the viability of sporozoites stored in liquid N₂ for 99 days. Counts of macroscopic focal lesions on the chorioallantois of embryos infected with E. tenella appears to be an accurate method for assessing the viability of sporozoites of this species.     

Immunity-related gene single nucleotide polymorphisms associated with Rhodococcus equi infection in foals

In previous work, we found significant associations of horse polymorphic microsatellite and immunity-related (IR) gene markers with Rhodococcus equi infection of foals. Here, a statistically significant association between a single nucleotide polymorphism (SNP) within the interleukin 7 receptor-encoding gene (IL7R) with high R. equi burden in transtracheal aspirates was found (Fisher’s F = 0.043, odds ratio: 8.00, 95% confidence interval: 1.127–56.795). Further positional and/or functional candidate genes investigated TLR2, IL13, IL17A, IL28R, TACE/ADAM 17 and GBP1, were not associated with infection in this study. SNPs analysed were found by sequencing and appropriate restriction fragment length polymorphism markers were developed. Their associations with R. equi infection were tested by genotyping thoroughbred foals from the original study. The association was confirmed by analysing genotypes composed with genes previously reported to be associated with R. equi infection in the same group.     

Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998

The horse-parasitizing species Babesia equi Laveran, 1901 was redescribed as Theileria equi Mehlhorn, Schein 1998 and, thus, transferred from one valid genus to another. This transfer was needed since it turned out that this horse parasite showed the relevant characteristics of theilerians with regard to biological data, morphological features, biochemical properties, and molecular biological relationships. Received: 18 December 1997 / Accepted: 15 January 1998     

Invasion of the intestinal tract by sporozoites of Eimeria coecicola and Eimeria intestinalis in naive and immune rabbits

Naive and immune specific-pathogen-free rabbits were inoculated in the duodenum with sporocysts of Eimeria coecicola or Eimeria intestinalis. Samples were taken from the following tissues: duodenum (site of penetration of sporozoites), ileum (specific target site of the endogenous development of E. intestinalis), vermiform appendix (target site of E. coecicola) and two extraintestinal sites, mesenteric lymph nodes (MLNs), and spleen. The presence of sporozoites was checked by immunohistochemistry. In rabbits primary-infected with E. coecicola, large numbers of sporozoites were detected in the duodenum, extraintestinal sites, and vermiform appendix. The abundance of sporozoites in the spleen, MLN, and appendix was significantly reduced in the immune rabbits, and the migration seemed impeded. In the rabbits infected with E. intestinalis, sporozoites were absent in the spleen and MLN, indicating that the route of migration is different from that of E. coecicola. The number of sporozoites in the crypts of the ileum was markedly reduced in the immune animals.     

Salivary gland extract from engorged Ixodes ricinus (Acari: Ixodidae) stimulates in vitro growth of Borrelia burgdorferi sensu lato

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK‐H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Use of the polymerase chain reaction for identification and quantification of Theileria parva protozoa in Rhipicephalus appendiculatus ticks

The polymerase chain reaction (PCR) was adapted for detection of Theileria parva sporoblasts in Rhipicephalus appendiculatus ticks by comparison with staining of histological preparations of ticks with methyl green and pyronin (MGP). Two 32mer primers (IL174 and IL179) were used to amplify Theileria parva (Muguga isolate) DNA from the TPR 1 region of the genome by the PCR. Detection of T. parva was carried out with dissected salivary glands and whole ticks preserved in ethanol. Adult ticks which fed as nymphs on a T. parva infected calf were used in three experiments. Firstly, 70 whole ticks divided into 7 batches representing the rising and falling parasitaemia of the calf were used to show that detection of infection by the PCR was significantly correlated with MGP staining. Secondly, 120 dissected ticks were used from 4 different batches representative of the overall infection profile within the ticks to show a high correlation between PCR quantification within tick salivary glands and MGP count data of the paired gland. Thirdly, 120 ticks were used in batches selected for high and low infections. Bloodmeal contaminants from partially fed adult ticks, present in 60 out of the 120 ticks used, did not inhibit the PCR amplification of T. parva DNA. This experiment also showed a great increase in infection detection in partially fed batches of ticks compared to the untreated batches. Received: 5 August 1996 / Accepted: 15 October 1996     

Comparative efficacy of Annona squamosa and Azadirachta indica extracts against Boophilus microplus Izatnagar isolate

In the search of developing herbal acaricides, eight medicinal plants were screened for their efficacy against Boophilus microplus, the widely distributed tick species in India. Of the seven extracts screened, the extracts prepared from the Annona squamosa seed showed very high level of efficacy (70.8%) after 24 h of treatment. The effect of treatment on oviposition of the survived ticks was also assessed, and a significant reduction (P < 0.05) in the reproductive index was noted in comparison to control. When efficacy of the in vitro optimized concentration of A. squamosa was compared with previously tested extract of Azadirachta indica in in vivo model, it was observed that the extracts prepared from A. indica is more efficacious than the extracts of A. squamosa. A comparable efficacy against B. microplus fed on animals treated with herbal extracts and commonly used synthetic acaricide was noted. The possibility of using the herbal extracts in IPM format for the management of ticks is discussed.     

Immunoglobulin G Subisotype Responses of Pneumonic and Healthy, Exposed Foals and Adult Horses to Rhodococcus equi Virulence-Associated Proteins

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals and in immunocompromised humans. Replication of virulent isolates within macrophages correlates with the presence of a large plasmid which encodes a family of seven virulence-associated proteins (VapA and VapC to VapH), whose functions are unknown. Although cell-mediated immunity is thought to be crucial in eliminating R. equi infection, antibody partially protects foals. The antibody response to both VapA and VapC was similar in six adult horses and six naturally exposed but healthy foals, as well as in eight foals with R. equi pneumonia. The immunoglobulin G (IgG) subisotype response of pneumonic foals to Vap proteins was significantly IgGb biased and also had a trend toward higher IgGT association compared to the isotype association of antibody in adult horses and healthy exposed foals. This suggests that in horses, IgGb and IgGT are Th2 isotypes and IgGa is a Th1 isotype. Furthermore, it suggests that foals which develop R. equi pneumonia have a Th2-biased, ineffective immune response whereas foals which become immune develop a Th1-biased immune response. Pneumonic foals had significantly more antibody to VapD and VapE than did healthy exposed foals. This may indicate a difference in the expression of these two Vap proteins during persistent infection. Alternatively, in pneumonic foals the deviation of the immune response toward VapD and VapE may reflect a bias unfavorable to R. equi resistance. These data indicate possible age-related differences in the equine immune response affecting Th1-Th2 bias as well as antibody specificity bias, which together favor the susceptibility of foals to R. equi pneumonia.