Production and characterization of monoclonal antibodies against midgut of ixodid tick,Haemaphysalis longicornis

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.     

Molecular cloning of two caspase-like genes from the hard tick Haemaphysalis longicornis

We identified two caspase-like genes from the midgut cDNA library of the hard tick, Haemaphysalis longicornis. Sequence analysis showed that these cDNAs encoded homologues of caspase-2 and caspase-8 that were categorized as apoptosis initiators. The H. longicornis caspase-2 (Hlcaspase-2) cDNA encodes 340 amino acid residues with a predicted molecular weight (Mw) of 38.5 kDa. Another cDNA identified as the H. longicornis caspase-8 (Hlcaspase-8) encodes 306 amino acid residues with an estimated Mw of 35.3 kDa. A catalytic active site was highly conserved in Hlcaspase-8 but not in Hlcaspase-2. RT-PCR analysis showed that both Hlcaspase-2 and Hlcaspase-8 were expressed in tick midgut and salivary glands. This is the first report of the molecular cloning of apoptosis-related genes in the tick.     

Connective tissue growth factor expression in the rat remnant kidney model and association with tubular epithelial cells undergoing transdifferentiation

Connective tissue growth factor (CTGF) has been shown to mediate many actions of transforming growth factor-beta (TGF-beta) in the fibrotic response in several diseases. We compared expression of CTGF, TGF-beta, platelet-derived growth factor (PDGF), TNF-alpha, and interleukin-1 (IL-1) by in situ hybridization in Sprague-Dawley rats euthanized at 0, 2, 4, and 8 weeks after 5/6 nephrectomy using the rat remnant kidney model of renal failure. Collagen was evaluated by trichrome stains, immunohistochemistry, and electron microscopy. We compared expression patterns to cells undergoing metaplasia. Tubular epithelial regeneration and transdifferentiation to myofibroblasts were assessed morphologically and by proliferating cell nuclear antigen, smooth muscle actin, desmin, and vimentin immunohistochemistry. CTGF expression was minimal in controls, mild at 2 weeks and marked by 4 to 8 weeks in interstitial fibroblasts, coinciding with damage, regeneration, and fibrosis. TGF-beta expression was increased in many cell types at 2 weeks, increased further by 4 weeks, then remained constant. PDGF-B messenger RNA was found in many stromal cells at 2-4 weeks, but expression decreased at 8 weeks. No significant IL-1 or TNF-alpha staining was detected. We conclude that CTGF and interacting factors are associated with development or progression of chronic interstitial fibrosis. Proximity of CTGF, TGF-beta, and PDGF mRNA expression to regenerative epithelial cells and those transdifferentiating to myofibroblasts suggests that growth factors may modulate renal tubular epithelial differentiation.     

Development of Babesia gibsoni in the midgut of larval tick, Rhipicephalus sanguineus.

Studies were made on the development of Babesia gibsoni in the midgut of the larval tick, Rhipicephalus sanguineus. Six hr after repletion, merozoites of B. gibsoni, freed from erythrocytes, were observed in the midgut contents of the tick. After that, within 24 hr, those merozoites were transformed into the ring-forms which were relatively large, 2-3 microns in diameter. Later, the ring forms developed into the spherical forms which were subelliptical in shape and 4-6 microns in diameter. Within 2-4 days, the elongated forms, 5-8 microns in length, were found. At this time, some of the binucleated fusion form has assumed a form intermediate between the spherical and elongated-forms. About 5-6 days after repletion, large round or elliptic zygotes, 8-10 microns in diameter, were observed in the tick gut.     

Passive immunization with monoclonal antibodies: effects on Haemaphysalis longicornis tick infestation of BALB/c mice

Tick vaccine development plays an important role in current tick control strategies. Previously, we have produced three different isotypes of monoclonal antibodies (mAbs) which recognized a midgut protein of adult Haemaphysalis longicornis. These mAbs, typed as IgG1, 2a, and 2b, reacted with a 76 kDa surface protein of midgut cells. We speculated that the 76 kDa protein may be an unknown antigen for a tick vaccine and the three mAbs may work as probes to clone the protein. In this study, to test whether these three isotypes have anti-tick effects and if so which works more effectively, we conducted passive immunization in BALB/c mice with each of the mAbs, and infested the mice with adult ticks. All isotypes significantly reduced the number of hatched larvae, compared to controls, however, no differences in the magnitude of the reduction were observed among the three.     

铁蛋白1基因在边缘革蜱各发育龄期、吸血期及不同器官中的表达分析

试验旨在了解铁蛋白1（ferritin 1,Fer1）基因在边缘革蜱各发育龄期、吸血期及不同器官中的表达情况。用实时定量荧光PCR法分析了Fer1基因在边缘革蜱各发育阶段、吸血期和不同器官中的相对表达情况,以饥饿雌蜱为基准（1 FC）,用2^-△△Ct法计算结果。结果显示,雌性蜱在开始吸血后第72 h Fer1基因的相对表达量最高,在刚刚孵化的饥饿成蜱（雌、雄）、卵及幼蜱中相对表达量较低;在若蜱中相对表达量较高,其中,幼蜱和若蜱在饥饿状态下比同时期饱血蜱的相对表达量高。在雌蜱的不同器官中,以饱血蜱唾液腺为基准（1 FC）,半饱血蜱中肠的表达量最高,在卵巢和马氏管不表达;在饱血蜱唾液腺、中肠、卵巢和马氏管中均表达,其中,在卵巢和马氏管中的表达量较高,中肠次之,唾液腺中的表达较低。试验结果表明,边缘革蜱体内Fer1基因的相对表达量随着蜱吸血过程变化而变化,这与蜱体内消化细胞的完善及消化酶在该过程中的变化相关,可为该类蜱铁代谢通路中关键功能基因的研究提供参考。     

Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis

Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoretic separation of midgut membrane proteins was greatly improved by using liquid-phase isoelectric focusing combined with one-dimensional or two-dimensional (2-D) gel electrophoresis. A selection of differentially expressed proteins were subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the identified Babesia-affected tick midgut proteins were six proteins that are implicated in signaling processes, including three Ca(2+)-binding proteins, a guanine nucleotide-binding protein, a protein with signal peptide activity and a translocon-associated receptor protein. Up-regulation of five metabolic enzymes indicated parasite-induced changes in electron and proton transport, protein processing and retinoic acid metabolism. Among the down-regulated proteins were a molecular chaperone, a cytoskeletal protein and a multifunctional protein of the prohibitin family. Identification of these proteins may provide new insights into the molecular interactions between B. bovis and its tick vector, and could lead to identification of anti-tick and transmission-blocking vaccine candidates.     

Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures.

Insulin-like growth factor-binding proteins (IGFBP) regulate the biological functions of insulin-like growth factors (IGF) and may affect cell growth through IGF-independent actions. Growth factors and hormones have been shown to alter IGFBP production by target cells suggesting that the effects of these factors may be partially mediated by the local production of IGFBP. Growth factors, including IGF-I, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) have potent effects on satellite cell proliferation and differentiation, and some of these factors have been shown to alter IGFBP production in various cell types. Consequently, some of their actions on muscle satellite cells may be mediated by the local production of IGFBP. In this study, we measured the effects of IGF-I, bFGF, and TGF-beta1 on IGFBP production by primary porcine satellite cell (PSC) cultures after first determining physiologically active concentrations of these growth factors to use according to [3H]thymidine incorporation dose responses. There is little information on the effects of these growth factors on IGFBP production in primary porcine myogenic cells due to the confounding affects of contaminating nonmuscle fibroblasts. Comparative studies show that primary porcine satellite cells produce IGFBP-3 and -5 whereas porcine muscle-derived nonfusing cells (FIB) produce IGFBP-2 and -4 but not IGFBP-3 or -5. Because of this, our investigations have focused on growth factor-induced production of IGFBP-3 and -5 in primary porcine satellite cells cultures. Both IGF-I and bFGF exhibited dose-dependent increases in [3H]thymidine incorporation with increasing concentration from 1 to 50 ng/mL (P < 0.05), whereas TGF-beta1 caused a dose-dependent decrease from 0.01 to 0.5 ng/mL (P < 0.05). When 20 ng/ mL of IGF-I was added to the media, IGFBP-3 was increased approximately 65% (P < 0.05) and IGFBP-5 was increased approximately twofold (P < 0.05). The addition of 0.5 ng/mL TGF-beta1 caused more than a two-fold increase in IGFBP-3 (P < 0.05) and approximately an 80% increase in IGFBP-5 (P < 0.05), whereas 50 ng/ mL of bFGF caused approximately 40% (P < 0.05) and 70% (P < 0.05) increases in IGFBP-3 and -5, respectively. Neither IGFBP-3 nor -5 was detectable in the conditioned media from fibroblasts whether or not IGF-I, TGF- beta1 or bFGF were present. These data suggest that the effects of IGF-I, TGF- beta1 and bFGF on porcine satellite cells may in part be through the autocrine/ paracrine production of IGFBP-3 and -5 by porcine satellite cells.     

Immunohistochemical analysis of molecular events in tubulo-interstitial fibrosis in a mouse model of diffuse mesangial sclerosis (ICGN strain)

Diffuse mesangial sclerosis (DMS) is one of the hereditary glomerular diseases and histologically characterized by severe glomerulosclerosis and subsequent tubulo-interstitial fibrosis (TIF). In DMS patients, renal dysfunction correlates well with TIF, rather than with glomerular lesions. Thus, molecular mechanisms whereby TIF in DMS progresses should be addressed. Previously, we found that nephrotic ICGN mice manifest DMS-like lesions and develop renal dysfunction in accordance with onset of TIF. In the present study, we investigated fibrogenic events involved in the progression of TIF after DMS manifestation, using the DMS mouse model. Immunohistochemistry revealed that expression of transforming growth factor-beta (TGF-beta) was rare in the interstitial cells of the nephrotic mice at the early-stage of DMS, while the TGF-beta expression became evident in the late-stage DMS mice. Platelet-derived growth factor (PDGF) was mildly expressed in the distal tubules of the early-stage DMS mice, whereas the PDGF expression markedly increased at the late-stage of DMS. As a result, alpha-actin-positive myofibroblastic cells were found dominant in the interstitial spaces of the late-stage DMS mice. Finally, TIF became severe in accordance with the overexpressions of these molecules. Our results suggest that in our murine model: 1) persistent proteinuria leads to over-expression of TGF-beta and PDGF in non-glomerular areas; 2) these cytokines provoke interstitial myofibroblast accumulation; and 3) the myofibroblasts produce fibrotic matrix proteins in the interstitial spaces. This process may possibly contribute to the development of TIF in DMS patients.     

Anaplasma marginale in tick cell culture

Anaplasma marginale was propagated in a tick cell line derived from Dermacentor variabilis embryos. The rickettsial organism was identified and monitored in culture by transmission electron microscopy and the indirect immunofluorescence technique, using specific monoclonal antibodies. Inoculation of the embryonic tick cell line with midguts of infected adult ticks (culture 1), nymphal ticks (culture 2) and adult ticks that were infected as nymphs and dissected as adults (culture 3) resulted in 3 continuous cultures of A marginale. Culture 1 had been maintained through 22 passages over a 11-month period; cultures 2 and 3 had been maintained for 18 passages over a 9-month period. Growth of A marginale in the cell line began in the area of the nuclear membrane at approximately 4 days after inoculation or transfer. Thereafter, the organisms were observed in inclusions scattered throughout the cytoplasm of the host cells. Maximal growth of the organism occurred at 7 to 14 days, after which numbers of inclusions rapidly decreased to minimal or undetectable levels. The organism began new cycles of growth with each 1:5 to 1:10 split and transfer of the host cells. Electron microscopy of recently infected cells revealed a morphology of the organism that closely resembled that observed in marginal bodies of infected erythrocytes. After several passages, A marginale organisms had a varied morphology and resembled the organism described in midgut cells of naturally infected ticks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinctive staining of colonies of Cowdria ruminantium in midguts of Amblyomma hebraeum

Mallory's phloxine-methylene blue stain was used to differentiate colonies of Cowdria ruminantium in midgut epithelial cells of nymphal Amblyomma hebraeum that had been infected as larvae. Gut tissues were collected from nymphs that had fed on a susceptible sheep and were fixed in formol-saline on the day of repletion. Paraffin sections, 3-4 micron thick, were then stained and this rendered colonies and cell nuclei densely blue against a uniformly pink background of tick tissues. Colonies were easily distinguished from nuclei by their specific morphology. This method of parasite visualization may be adapted to field-collected ticks for rapid detection of C. ruminantium or to assays of susceptibility of tick populations to various strains of the organism.     

Resistance by the blue tick (Boophilus decoloratus) to the synthetic pyrethroid, fenvalerate

The Shaw larval test, in conjunction with adult tick immersion and stall tests, was utilized to confirm that a field strain of B. decoloratus, from Natal, is highly resistant to the ixodicide fenvalerate (Factor of resistance 4,744). This resistance developed over a reported 18 months of usage for cattle dipping.     

Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum

The development of colonies of Cowdria ruminantium was studied in midgut epithelial cells of adult Amblyomma variegatum that had become infected by feeding as nymphs on cattle with experimentally induced heart-water disease. Colonies were not observed in gut tissues obtained from nymphs during the feeding period, but were present in midgut epithelial cells of ticks obtained at 15 days after they were replete through molting to the adult stage. Colonies were small (1 to 10 micron) initially, but as tick development progressed, their diameter increased to as much as 60 micron. With electron microscopy, colonies were observed to be membrane bound and contained pleomorphic organisms that were reticulated. The organisms seemed to be dividing by binary fission. Many colonies contained a large, electron-dense inclusion that was morphologically similar to hemoglobin deposits found in the cytoplasm of midgut epithelial cells of recently fed ticks. Cowdria ruminantium was often observed adhered to these inclusions.     

中华硬蜱叮咬蜱抗原免疫接种宿主后中肠组织化学的动态观察

选用中华硬蜱叮咬中肠抗原免疫接种组及初次叮咬组和佐剂对照组宿主并分别在不同时间(叮咬24、48、72h以及5、8d)对蜱中肠蛋白质、糖原及多糖进行了动态观察,并对消化细胞核DNA含量进行测定。结果显示:蜱叮咬中肠抗原免疫接种宿主后可使中肠上皮发生破坏,消化细胞破损,基膜可出现松散和断裂,消化细胞蛋白质含量较初次叮咬组和佐剂对照组宿主明显降低,而糖原及多糖含量基本不变。消化细胞DNA在叮咬无免疫力宿主(初次叮咬组和佐剂对照组)早期24h主要为2倍体,随着吸血量增加、细胞核倍体数加大,第5天达7.06倍体,而叮咬中肠抗原免疫接种组宿主后,消化细胞细胞核倍体数增加不明显,与叮咬无免疫力宿主有显著性差异(P>0.01),提示中华硬蜱叮咬中肠抗原免疫接种宿主后,可干扰中肠消化细胞蛋白质和核酸(DNA)的合成代谢,为进一步揭示抗蜱免疫机理提供了理论依据。     

Stimulus-dependent release of tissue-regenerating factors by equine platelets

Reasons for performing the study: Platelet‐rich plasma (PRP) is increasingly used for treatment of orthopaedic injuries. However, the effects of different stimuli on the release pattern of regenerative and proinflammatory factors from equine platelets are largely unknown and an optimal treatment protocol remains to be established. Objectives: The aim of this study was to identify a stimulus that enhanced release of histopromotive factors (platelet‐derived growth factor BB [PDGF] and transforming growth factor 1β[TGF]) without causing concurrent release of a proinflammatory mediator (CCL5). Methods: Washed platelets were prepared from 6 healthy ponies and release of growth factors and CCL5 measured using commercially available ELISAs for human proteins following incubation with or without thrombin, chitosan or equine recombinant tumour necrosis factor (erTNF) over 24 h and subsequently over 96 h. Additionally, noncoagulated samples were analysed. Results: Regardless of whether a stimulus was present or what stimulus was used, PDGF and TGF release was maximal by 0.5–1 h when clot formation took place and very little release was observed after 24 h. Growth factor release was minimal in noncoagulated samples. In contrast, CCL5 release was not associated with coagulation and appeared to persist for much longer. High concentrations of erTNF caused significantly greater release of CCL5 at 6 h than any other stimulus tested. Conclusions: Growth factor release from equine platelets is dependent on coagulation but independent of the initiating stimulus, and is accompanied by more sustained release of proinflammatory mediators. Potential relevance: Supernatants collected from coagulated platelets could be an alternative treatment to PRP.     

Variations of plasmatic concentrations of Insulin-like Growth Factor-I in post-pubescent horses affected with developmental osteochondral lesions

Developmental osteochondral lesions are often encountered in the equine population and are a major cause of lameness. Different growth factors that act systemically as well as locally regulate the growth of cartilage. Among them is Insulin-like Growth Factor I that has been demonstrated to promote chondrocyte growth and differentiation and that has been shown to influence cartilage repair. The aims of this study were to investigate differences in circulating plasma levels of Insulin-like Growth Factor-I in post-pubescent horses affected with developmental osteochondral lesions compared to unaffected ones. Significantly higher values of circulating Insulin-like Growth Factor-I levels were found in the affected group (n = 82) compared to controls (n = 86). This result may still reflect an earlier imbalance in IGF-I levels from horses with developmental osteochondral lesions considering the aetiopathological link which has been made between IGF-I and the occurrence of osteochondrosis. However, other studies have shown increased expression of IGF-I after cartilage damage. The higher levels found in this study could be due to a healing response of the cartilage to the damage caused by the osteochondral lesions.     

Detection of colonies of Anaplasma marginale in salivary glands of three Dermacentor spp infected as nymphs or adults

Salivary glands from males of 3 Dermacentor species (D andersoni, D variabilis and D occidentalis) that were infected with either the Virginia or Idaho isolate of Anaplasma marginale as nymphs or adults were examined for colonies of A marginale by use of light and electron microscopy. Prior to dissection of salivary glands, exposed ticks were held at 25 C for 15 to 18 days, followed by a 3-day incubation at 37 C. Ticks of 2 species transmitted A marginale to calves; the third tick species was confirmed infected by demonstration of typical colonies in tick gut cells, but transmission was not attempted; Colonies of A marginale were seen with light microscopy in salivary glands of all 3 species of ticks; they were located in acinar cells that contained simple granules. Colonies varied morphologically from small, compact ones to larger structures that contained distinct organisms and often were adjacent to the host cell nucleus. Electron microscopy confirmed that the colonies were rickettsial organisms. Morphologic features of A marginale varied and included reticulated forms, forms with electron-dense centers, and small particles; these various forms were similar to those described previously in midgut epithelial cells of ticks. We believe that the organism seen within tick salivary glands may replicate in the glands before its transmission to the vertebrate host.     

Effects of the paralysis tick, Ixodes holocyclus, on the electrocardiogram of the Spectacled Flying Fox, Pteropus conspicillatus

OBJECTIVE: To evaluate cardiac electrical function in the Spectacled Flying Fox (bat) infested with Ixodes holocyclus. DESIGN: Prospective clinical investigation of bats treated for naturally occurring tick toxicity. PROCEDURE: ECGs were performed on bats with tick toxicity (n = 33), bats that recovered slowly (n = 5) and normally (n = 5) following treatment for tick toxicity, and on normal bats with no history of tick toxicity (n = 9). RESULTS: Bats with tick toxicity had significantly prolonged corrected QT intervals, bradycardia and rhythm disturbances which included sinus bradydysrhythmia, atrial standstill, ventricular premature complexes, and idioventricular bradydysrhythmia. CONCLUSIONS: The QT prolongation observed on ECG traces of bats with tick toxicity reflected delayed ventricular repolarisation and predisposed to polymorphic ventricular tachycardia and sudden cardiac death in response to sympathetic stimulation. The inability to document ventricular tachycardia in bats shortly before death from tick toxicity may be explained by a lack of sympathetic responsiveness attributable to the unique parasympathetic innervation of the bat heart, or hypothermia-induced catecholamine receptor down-regulation. Bradycardia and rhythm disturbances may be attributable to hypothermia.     

Purification and characterization of a 45-kDa concealed antigen from the midgut membranes of Ornithodoros erraticus that induces lethal anti-tick immune responses in pigs

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In the search for a vaccine against this parasite, a crude extract of tick midgut membranes (GME) was obtained that in pigs and mice induced a protective response able to kill up to 80% of the nymphs in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. To identify the protective antigens, the GME was subjected to successive biochemical fractionations and the resulting simpler protein fractions were inoculated in pigs. A 45-kDa antigen, the so-called Oe45, was detected, purified and demonstrated to be responsible for the protection induced by the GME. Oe45 seems to be a membrane protein that is presumably expressed on the luminal membrane of midgut epithelial cells. Oe45 consists of at least two differently charged bands (cationic and neutral), which show antigenic cross-reactivity. The possibility that these bands might be different isoforms of the same protein is discussed. Although Oe45 is constitutively expressed at low levels throughout the trophogonic cycle, its expression is up-regulated by the ingestion of blood, as suggested by the higher levels observed between 6 and 72 h post-feeding.