Failure of the Amblyomma cajennense nymph to become infected by Theileria equi after feeding on acute or chronically infected horses

Tick-borne diseases in horses are caused by the intraerythrocytic protozoan parasites Theileria equi and Babesia caballi. Although T. equi is highly endemic in Latin America, the New World vector of this important parasite is controversial. The aim of this study was to test the ability of nymph Amblyomma cajennense ticks acquire infection by T. equi following feeding on infected horses. Three experiments were performed: tick acquisition of T. equi from an experimentally infected horse, tick acquisition of T. equi from naturally infected foals and tick acquisition of T. equi from a chronically infected horse. A. cajennense adults were dissected and salivary glands were collected in aliquots. Methyl green pyronin staining of the salivary glands did not show the presence of hypertrophy of acini or cell nuclei normally suggestive of Theileria spp. infection. The pools of salivary glands were negative for Theileria DNA in nested PCR assays. Histopathological analysis failed to detect sporoblast and sporozoites of T. equi in salivary gland acini. This study was not able to observe infection of the A. cajennense by T. equi.     

Seroprevalence and Potential Risk Factors Associated with Neospora spp. Infection among Asymptomatic Horses in Jordan

This study aimed to determine the seroprevalence and to identify risk factors associated with Neospora spp. infection in horses in Jordan. Management related data were collected from each farm and individual horses. Sera from 227 horses from 5 of 6 climatic regions in Jordan were analyzed for the presence of antibodies to Neospora spp. by ELISA kit. The study was performed during spring of 2010. The association between seropositivity and risk factors was analyzed. A total of 7 (3%) of 227 sera had antibodies for Neospora spp. There was a significant regional difference (P=0.018) between the 5 climatic regions. Positive cases were located in Amman and Irbid, while the other regions (Zarqa, Jordan Valley, and Wadi Mousa) had zero prevalence. The use of anthelmintics at least once a year resulted in a significant reduction of the seroprevalence to Neospora spp. (1.6% vs 9.8%). However, this might be a phenomenon by chance and a better hygiene since owners can invest in anthelmintics. Other risk factors such as age, gender, breed, usage, body condition score, grazing, presence of other animals mixed with the horses in the same property, and a history of previous diseases were not significantly associated with the seroprevalence to Neospora spp. infection. This is the first study to report on the presence of Neospora seropositive horses in Jordan. Further studies are warranted to better understand the role of certain risk factors in the transmission of Neospora spp. among horse population and to determine which Neospora spp. are responsible for the infection.     

Salivary calculi microbiota: new insights into microbial networks and pathogens reservoir

Sialolithiasis represents the most common disorders of salivary glands in middle-aged patients. It has been hypothesized that the retrograde migration of bacteria from the oral cavity to gland ducts may facilitate the formation of stones. Thus, in the present study, a microbiome characterization of salivary calculi was performed to evaluate the abundance and the potential correlations between microorganisms constituting the salivary calculi microbiota. Our data supported the presence of a core microbiota of sialoliths constituted principally by Streptococcus spp., Fusobacterium spp. and Eikenella spp., along with the presence of important pathogens commonly involved in infective sialoadenitis.     

First detection of anti-Besnoitia spp. specific antibodies in horses and donkeys in Italy

Among Apicomplexa protozoa infecting equids, Besnoitia spp., Toxoplasma gondii and Neospora spp. represent important issues from a sanitary and zootechnical viewpoint. However, only scarce epidemiological data are available on the spread of the infections in horses and donkeys in Europe. Therefore, a serosurvey was planned to estimate the prevalence of these Sarcocystidae species in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory WB was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. The study confirmed the circulation of Besnoitia spp. among equids in Europe. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. Nevertheless, it is noteworthy to consider that consumption of horse meat could represent a source for human toxoplasmosis.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

Biochemical characterization of α- and β-glucosidases in alimentary canal, salivary glands and haemolymph of the rice green caterpillar, Naranga aenescens M. (Lepidoptera: Noctuidae)

The biochemical properties of ??- and ??-glucosidase in salivary glands, alimentary canal and haemolymph of Naranga aenescens larvae, one of the most damaging pests of the rice crop in Iran, were investigated. The specific activity of ??-glucosidases were 3.88, 2.74 and 1.58 ??mol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The specific activity of ??-glucosidases were 1.27, 0.077 and 0.414 ??mol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The optimal pH for ??-glucosidases were 6.0, 6.0?C8.0 and 6.0 and the maximum activity for ??-glucosidases were obtained at pH 6.0, 5.0?C7.0 and 5.0 in alimentary canal, salivary glands and haemolymph, respectively. The optimum temperatures for ??-glucosidases were determined at 55°C in alimentary canal, 35?C45°C in salivary glands and 55°C in haemolymph, whereas the ??-glucosidases reached their optimum at 45°C in all three tissues. Effect of metal ions on the activity of ??- and ??-glucosidases showed that K⁺ (20 mM) and Mg²⁺ (10 and 20 mM) increased N. aenescens ??- and ??-glucosidases activities from salivary glands, while Ca²⁺ increased ??- and ??-glucosidases activities in haemolymph. In the presence of Fe²⁺, Mn²⁺, Hg⁺ and Zn²⁺ (10, 20 mM) and Hg²⁺ (20 mM), these enzymes from all tissues were completely inactivated. K _m values were estimated for the ??-glucosidases as 3.96, 0.547 and 3.084 mM and for ??-glucosidases as 1.93, 1.014 and 1.93 mM in the alimentary canal, salivary gland and haemolymph, respectively. The zymogram analyses of N. aenescens crude extracts indicated the presence of at least two isoforms for ??-glucosidase and one isoform for ??-glucosidase.     

Two ligands signal through the Drosophila PDGF/VEGF receptor to ensure proper salivary gland positioning

The Drosophila embryonic salivary gland is a migrating tissue that undergoes a stereotypic pattern of migration into the embryo. We demonstrate that the migratory path of the salivary gland requires the PDGF/VEGF pathway. The PDGF/VEGF receptor, Pvr, is strongly expressed in the salivary glands, and Pvr mutations cause abnormal ventral curving of the glands, suggesting that Pvr is involved in gland migration. Although the Pvr ligands, Pvf1 and Pvf2, have distinct expression patterns in the Drosophila embryo, mutations for either one of the ligands result in salivary gland migration defects similar to those seen in embryos that lack Pvr. Rescue experiments indicate that the PDGF/VEGF pathway functions autonomously in the salivary gland. The results of this study demonstrate that the Drosophila PDGF/VEGF pathway is essential for proper positioning of the salivary glands.     

The use of a novel combination of diagnostic molecular and cytogenetic approaches in horses with sexual karyotype abnormalities: A rare case with an abnormal cellular chimerism

Sex chromosome aberrations are known to cause congenital abnormalities and unexplained infertility in horses. Most of these anomalies remain undiagnosed because of the complexity of the horse karyotype and the lack of specialized laboratories that can perform such diagnoses. On the other hand, the utilization of microsatellite markers is a technique widely spread in horse breeding, mostly because of their usage in parentage tests. We studied the usage of a novel combination of diagnostic approaches in the evaluation of a very uncommon case of chromosomal abnormalities in a Spanish purebred colt, primarily detected using a commercial panel of short tandem repeat (STR) makers. Based on these results, we performed a full cytogenetic analysis using conventional and fluorescent in situ hybridization techniques with individual Equus caballus chromosome X and Equus caballus chromosome Y painting probes. We also tested the presence of two genes associated with the sexual development in horses and an extra novel panel of eight microsatellite markers specifically located in the sex chromosome pair. This is the first case report of a leukocyte chimerism between chromosomally normal (64,XY) and abnormal (63,X0) cell lines in horses. Our results indicate that the use of the short tandem repeat markers as a screening technique and as a confirmation utilizing cytogenetic techniques can be used as a very interesting, easy, and nonexpensive diagnostic approach to detect chromosomal abnormalities in the domestic horse.     

Identification, expression and characterisation of a major salivary allergen (Cul s 1) of the biting midge Culicoides sonorensis relevant for summer eczema in horses

Salivary proteins of Culicoides biting midges are thought to play a key role in summer eczema (SE), a seasonal recurrent allergic dermatitis in horses. The present study describes the identification, expression and clinical relevance of a candidate allergen of the North American midge Culicoides sonorensis. Immunoblot analysis of midge saliva revealed a 66 kDa protein (Cul s 1) that was bound by IgE from several SE-affected (SE+) horses. Further characterisation by fragmentation, mass spectrometry and bioinformatics identified Cul s 1 as maltase, an enzyme involved in sugar meal digestion. A cDNA encoding Cul s 1 was isolated and expressed as a polyhistidine-tagged fusion protein in a baculovirus/insect cell expression system. The clinical relevance of the affinity-purified recombinant Cul s 1 (rCul s 1) was investigated by immunoblotting, histamine release testing (HRT) and intradermal testing (IDT) in eight SE+ and eight control horses. Seven SE+ horses had rCul s 1-specific IgE, whereas only one control animal had IgE directed against this allergen. Furthermore, the HRT showed rCul s 1 induced basophil degranulation in samples from seven of eight SE+ horses but in none of the control animals. rCul s 1 also induced immediate (7/8), late-phase (8/8) and delayed (1/8) skin reactivity in IDT on all SE+ horses that had a positive test with the whole body extract (WBE) of C. sonorensis. None of the control horses showed immediate or delayed skin reactivity with rCul s 1, and only one control horse had a positive late-phase response, while several non-specific late-phase reactions were observed with the insect WBE. Thus, we believe rCul s 1 is the first specific salivary allergen of C. sonorensis to be described that promises to advance both in vitro and in vivo diagnosis and may contribute to the development of immunotherapy for SE in horses.     

Chew and spit: tree-feeding notodontid caterpillars anoint girdles with saliva

Caterpillars of the notodontid Oedemasia leptinoides (formerly Schizura) use their mandibles to cut shallow girdles that encircle the petioles and stems of tree hosts. When girdles are complete, the larvae bathe the girdle surface with fluid. We test whether the fluid originates from the labial salivary glands or ventral eversible gland by blocking the openings to the glands and observing whether fluid is still released onto the girdles. Only larvae with functional labial salivary glands anointed girdles with fluid. Analysis of girdle rinses for a prominent salivary enzyme, glucose oxidase, confirmed that larvae apply saliva and documented that application occurs primarily at the end of girdling. We propose that girdling by notodontids, together with related furrowing and leaf-clipping behaviors exhibited by diverse caterpillar groups, serve at least in part to introduce salivary components to exposed vascular tissues; these compounds presumably function to suppress plant defensive responses normally elicited by caterpillar feeding.     

Respiration of larval salivary glands of Drosophila in relation to the activity of specific genome loci

Oxygen consumption of intact larval salivary glands of Drosophila hydei was measured after the addition of intermediates of the citric acid cycle or amino acids to the incubation medium. The effect of these substances on respiration of glands previously submitted to anaerobiosis in vivo was compared with that of glands of control larvae. Only isocitrate and tyrosine stimulated respiration of anaerobically treated glands to a much higher extent than glands of control larvae. This stimulatory effect was abolished when RNA or protein synthesis was inhibited. It is suggested that some of the specific puffs occurring as a consequence of anaerobiosis reflect gene activity required for an increase in utilization of isocitrate and tyrosine for respiration under conditions of stress.     

Saliva,Salivary Gland,and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) ^1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick ^3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick''s enzootic cycle ^14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva ^9,16-19.As a tick feeds the host typically responds with a strong hemostatic and innate immune response ^11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding ^{3,11,20,21,23}. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens ^3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.     

Anatomy and ultrastructure of the salivary (prosomal) glands in unfed water mite larvae Piona carnea (C.L. Koch, 1836) (Acariformes: Pionidae)

Anatomy and ultrastructure of prosomal salivary glands in the unfed water mite larvae Piona carnea (C.L. Koch, 1836) were examined using serial semi-thin sections and transmission electron microscopy. Three pairs of alveolar salivary glands shown are termed lateral, ventro-lateral and medial in accordance with their spatial position. These glands belong to the podocephalic system and are situated on the common salivary duct from back to forth in the above mentioned sequence. The arrangement of the medial glands is unusual because they are situated one after another on the medial (axial) body line, therefore they are termed anterior and posterior medial glands. The secretory duct of the anterior medial gland mostly turns right, and the duct of the posterior gland turns left. The salivary glands are located in the body cavity partly inside the gnathosoma and in the idiosoma in front of the brain (synganglion). Each gland is represented by a single acinus (alveolus) and is composed of several cone shaped secretory cells arranged around the large central (intra-acinar) cavity with the secretory duct base. The cells of all glands are filled with secretory vesicles of different electron density. The remaining cell volume is occupied by elements of rough endoplasmic reticulum, and the membrane enveloping vesicles may have ribosomes on its external surface. Large nuclei provided with large nucleoli occupy the basal cell zones. The pronounced development of the prosomal salivary glands indicates their important role in extra-oral digestion of water mite larvae.     

Occurrence of Thelazia lacrymalis (Nematoda, Spirurida, Thelaziidae) in native horses in Italy

A survey on the prevalence of Thelazia spp. in the province of Bari (Apulia region, Italy) in slaughtered native horses was conducted from June 20, 1995 to April 3, 1996. Both eyes from 409 ten-month- to 4-year-old native animals were examined. Sixty horses (14.7%) were found parasitized by Thelazia lacrymalis. Three hundred-sixty one parasite specimens (220 females, 99 males and 42 larvae) were collected with a mean count burden of 6.0-5.1 (range 1 to 20) per head. T. lacrymalis specimens were found free in the conjunctiva and behind the nictitancte, in the excretory ducts of the Harderian gland and in the ducts of the lacrimal glands. Thelazia specimens were significantly more common in horses of 3-4 years. Gross examination of all infected animals showed a follicular conjunctivitis. This is the first report of T. lacrymalis horse infection in Italy.     

Serological Detection of Borrelia burgdorferi among Horses in Korea

Lyme disease is a tick-borne zoonotic infectious disease caused by Borrelia burgdorferi. The present study assessed the infection status of B. burgdorferi among horses reared in Korea using ELISA and PCR. Between 2009 and 2013, blood samples were collected from 727 horses throughout Korea. Data for each animal including age, gender, breed, and region of sample collection were used for epidemiological analysis. Overall, 38 (5.2%; true prevalence: 5.5%) of 727 horses were seropositive by ELISA. There were statistically significant differences according to breed and region (P<0.001) whose differences might be attributed to the ecology of vector ticks and climate conditions. Using 2 nested PCR, none of the samples tested positive for B. burgdorferi. Thus, a positive ELISA result can indicate only that the tested horse was previously exposed to B. burgdorferi, with no certainty over the time of exposure. Since global warming is likely to increase the abundance of ticks in Korea, continuous monitoring of tick-borne diseases in Korean horses is needed.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.