Water uptake in the cat flea Ctenocephalides felis (Pulicidae: Siphonaptera)

To counteract water loss due to excretion, cuticular transpiration and respiration, various groups of arthropods have developed mechanisms for active uptake of water vapor from unsaturated air. In this study, active uptake capabilities and water loss rates were examined in the various developmental stages of the cat flea, Ctenocephalides felis. To determine critical equilibrium humidity, the lowest relative humidity at which active water uptake can occur, pre-desiccated immature and adult fleas were placed in a series of humidity regimes ranging from 44 to 93% RH. Active uptake occurred in larval stages at relative humidities above 53% and in pre-pupae at 75-93% RH. Pupae and adults did not demonstrate active uptake at any humidity. Optimal uptake for larvae occurred between 20 and 30 degrees C. When placed over Drierite (<10% RH), larval and adult stages demonstrated a higher rate of water loss than pre-pupal and pupal stages. Active water uptake is necessary to ensure proper development of the larvae of C. felis. Active uptake ceases after the larval-pupal ecdysis and it appears that adults have lost the ability to actively uptake water.     

Advances in the control of Ctenocephalides felis (cat flea) on cats and dogs

Cat fleas are the most important ectoparasite of cats and dogs worldwide. During the past ten years, topical and oral applications of insecticides such as fipronil, imidacloprid, lufenuron and, most recently, selamectin have revolutionized cat-flea control. Recent studies show that these therapies eliminate the need to treat indoor and outdoor environments, and their use markedly reduces the severity and prevalence of flea allergic dermatitis. Surveys have yet to reveal the development of insecticide resistance to these chemical compounds. Extending the longevity of these effective host-targeted therapies should be a major goal of the veterinary community.     

Acquisition and excretion of Bartonella quintana by the cat flea,Ctenocephalides felis felis

Bartonella quintana is transmitted by the infected faeces of body lice. Recently, this bacterium was detected in cat fleas (Ctenocephalides felis) and in two humans with chronic adenopathy whose only risk factor was contact with cat fleas. In this study, a total of 960 C. felis were divided into 12 groups (2 control groups and 10 infected groups) each containing 80 fleas. The fleas were fed B. quintana‐inoculated human blood at different dilutions (≈3.6 × 10⁴ ? 8.4 × 10⁹ bacteria) for 4 days via an artificial membrane. Subsequently, all flea groups were fed uninfected blood until day 13 postinfection (dpi). On day 3 pi, B. quintana was detected with two specific genes by quantitative PCR in 60–100% of randomly chosen fleas per dilution: 52% (26/50) in the infected fleas in Trial 1 and 90% (45/50) of the fleas in Trial 2. B. quintana was also identified by molecular and culture assays in flea faeces. The average number of B. quintana as determined by qPCR decreased until the 11th dpi and was absent in both trials at the 13th dpi. Bacteria were localized only in the flea gastrointestinal gut by specific immunohistochemistry. Our results indicate that cat fleas can acquire B. quintana by feeding and release viable organisms into their faeces. Therefore, fleas may play a role as vectors of trench fever or other clinical manifestations that are caused by B. quintana. However, the biological role of C. felis in the transmission of B. quintana under natural conditions is yet to be defined.     

Consumption of flea faeces and eggs by larvae of the cat flea,Ctenocephalides felis

The effects of the consumption of flea faeces and non-viable eggs on larval development in the cat flea Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) were investigated. Only 13.3% of larvae developed into adults when fed a diet of male or female flea faeces alone; however, 90% of larvae developed into adults when fed on flea faeces supplemented with non-viable flea eggs. When fed with non-viable eggs alone, larvae did not develop into adults. Nevertheless, non-viable eggs may provide critical supplemental nutrients, lacking in flea faeces and required for larval development. None of the larvae fed on flea faeces or non-viable eggs alone formed a cocoon. A diet of flea faeces alone significantly extended the second as well as third larval stadia compared to larvae fed on diets containing non-viable eggs. It is suggested that the cannibalism of fertile eggs may limit population growth in the cat flea.     

Establishment of the cat flea (Ctenocephalides felis felis) on the ferret (Mustela putorius furo) and its control with imidacloprid

As the ferret, Mustela putorius furo L. (Carnivora: Mustelidae), is becoming increasingly popular as a pet animal and as it is susceptible to the cat-flea, Ctenocephalides felis felis Bouché (Siphonaptera: Pulicidae), an experimental model was established for evaluating insecticidal treatments on this host. A high establishment rate (76.7-91.8%) was recorded when 60 unfed adult C. felis were placed on ferrets. This provided an adequate infestation for chemotherapeutic evaluation without causing undue discomfort to the host. Twelve ferrets were allocated to two groups matched for sex and individual ability to sustain a flea population. One group was treated topically with an imidacloprid spot-on formulation at a dose rate of 10 mg/kg body-weight on Day 0. All ferrets were infested with C. felis on Days -1, 7, 14, 21 and 28, and flea counts were performed 8 and 24 h post-treatment and one day after each subsequent infestation. Fleas were removed at all but the 8 h count (when they were returned to their host). Flea burdens were reduced by 95.3% (P < 0.001) within 8 h of treatment and 100% efficacy was recorded at 24 h. At 1, 2, 3 and 4 weeks post-treatment, protection against re-infestation was 92.9% (P < 0.001), 55.7% (P < 0.02), 18.3% (NS) and 7.4% (NS), respectively. Thus, at this dose rate, imidacloprid gave excellent efficacy against a resident C. felis population and provided a high level of residual activity for at least one week after treatment.     

Identification and characterisation of the dopamine receptor II from the cat flea Ctenocephalides felis (CfDopRII)

G protein-coupled receptors (GPCRs) represent a protein family with a wide range of functions. Approximately 30% of human drug targets are GPCRs, illustrating their pharmaceutical relevance. In contrast, the knowledge about invertebrate GPCRs is limited and is mainly restricted to model organisms like Drosophila melanogaster and Caenorhabditis elegans. Especially in ectoparasites like ticks and fleas, only few GPCRs are characterised. From the cat flea Ctenocephalides felis, a relevant parasite of cats and dogs, no GPCRs are known so far. Thus, we performed a bioinformatic analysis of available insect GPCR sequences from the honeybee Apis mellifera, the mosquito Anopheles gambiae, the fruit fly Drosophila melanogaster and genomic sequences from insect species. Aim of this analysis was the identification of highly conserved GPCRs in order to clone orthologs of these candidates from Ctenocephalides felis. It was found that the dopamine receptor family revealed highest conservation levels and thus was chosen for further characterisation. In this work, the identification, full-length cloning and functional expression of the first GPCR from Ctenocephalides felis, the dopamine receptor II (CfDopRII), are described.     

High phylogenetic diversity of the cat flea (Ctenocephalides felis) at two mitochondrial DNA markers

The cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) (Bouché), is the most common flea species found on cats and dogs worldwide. We investigated the genetic identity of the cosmopolitan subspecies C. felis felis and evaluated diversity of cat fleas from Australia, Fiji, Thailand and Seychelles using mtDNA sequences from cytochrome c oxidase subunit I (cox1) and II (cox2) genes. Both cox1 and cox2 confirmed the high phylogenetic diversity and paraphyletic origin of C. felis felis. The African subspecies C. felis strongylus (Jordan) is nested within the paraphyletic C. felis felis. The south East Asian subspecies C. felis orientis (Jordan) is monophyletic and is supported by morphology. We confirm that Australian cat fleas belong to C. felis felis and show that in Australia they form two distinct phylogenetic clades, one common with fleas from Fiji. Using a barcoding approach, we recognize two putative species within C. felis (C. felis and C. orientis). Nucleotide diversity was higher in cox1 but COX2 outperformed COX1 in amino acid diversity. COX2 amino acid sequences resolve all phylogenetic clades and provide an additional phylogenetic signal. Both cox1 and cox2 resolved identical phylogeny and are suitable for population structure studies of Ctenocephalides species.     

2-Phenylimidazo[1,2-b]pyridazine derivatives highly active against Haemonchus contortus

A series of 2-phenylimidazo[1,2-b]pyridazine derivatives were synthesized and evaluated for their in vitro anthelmintic activity against Haemonchus contortus. The most active compounds had in vitro LD₉₉ values of 30 nM, which is comparable to that of the benchmark commercial nematocide, Ivermectin.     

Indicators of sexual maturation and regression in the female cat flea, Ctenocephalides felis

Differences in the salivary glands, mesenteron epithelium and reproductive organs of female cat fleas, Ctenocephalides felis Bouché (Siphonaptera: Pulicidae), are related to the degree of reproductive maturation or regression. Contrary to previous ideas, blue bodies in the ovarioles are degenerate oocyte nuclei and their presence denotes failure of ripening oocytes to reach full maturity. A distinction between true corpora lutea and pseudo-corpora lutea is established, the presence of the former indicates successful oviposition, and of the latter, failure to complete maturation of eggs. Accurate indicators of sexual maturation and reproductive success are of potential value in assessing relative suitability of various hosts for a given flea species and therefore in assessing the degree of host specificity among fleas.     

Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.     

Molecular characterisation of nicotinic acetylcholine receptor subunits from the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae)

As part of a program to monitor the susceptibility of cat flea populations to the insecticide imidacloprid we have examined the cat flea nicotinic acetylcholine receptor, the target site protein of the neonicotinoid group of insecticides. Seven nAChR subunits (six alpha-type and one beta-type) were identified in cat flea using a degenerate PCR-based strategy. Five of these were expressed in vitro by creating chimeras containing the N-terminal ligand-binding domain of the cat flea subunits and the C-terminal region of the Drosophila Dalpha2 (SAD) subunit. Two of the five chimeric subunits, Cfalpha1/Dalpha2 and Cfalpha3/Dalpha2, when co-expressed with rat beta2 in Drosophila S2 cells, showed high-affinity binding of both epibatidine (Kd=1.6+/-0.6 and 0.13+/-0.06nM, respectively), and imidacloprid (Ki=142+/-34 and 28.7+/-2.4nM, respectively). It is likely therefore that Cfalpha1 and Cfalpha3 contribute to nAChR populations in vivo that are sensitive to imidacloprid. The identification of cat flea nAChR subunits that have a high affinity for imidacloprid presents candidate genes in which to look for resistance-associated mutations if target-site resistance to imidacloprid arises in domestic pet flea populations.     

Computational study of dynamic motions and possibility of crossing between surfaces in 3-hydroxy-5-(1H-pyrrol-2-yl)-2H-pyrrol-2-one

Potential energy surfaces (PESs) for tautomerism and two dynamic motions of 3-hydroxy-5-(1H-pyrrol-2-yl)-2H-pyrrol-2-one and its tautomer were calculated using density functional theory. Calculated energies confirm that T1 is 10.95 kJ mol^? 1 more stable than T2. Dynamic study of possible motions shows the high energy-level transition state at D8 = 90° for ring rotation and at D15 = 80° and 90° for rotation of OH bond, respectively, in T1 and T2. In addition, calculated rate constant for conversion of T1 to T2 (tautomerism) is 116 M^? 1 s^? 1, for relative rotations of rings in T1 and T2 are, respectively, 5.62 × 10^? 2 and 1.53 M^? 1 s^? 1 and for rotations of OH bond in T1 and T2 are, respectively, 3.56 × 10⁵ and 6.15 × 10⁴ M^? 1 s^? 1. In the next part of the study, orbital occupancies, natural bond orbitals (NBO) charges and hybridisation in relative rotation of rings and internal reaction coordinate (IRC) steps have been extracted to study the possibility of level crossing. These data show that PESs for IRC and ring rotation have different symmetries. So that these two potential curves cannot have effective non-adiabatic level crossing. However, the levels are weakly avoided or the possibility of level crossing is higher than related system because the energy difference between their barrier energies is not very high.     

Synthesis and biological activity of 2-anilino-4-(1H-pyrrol-3-yl) pyrimidine CDK inhibitors

A series of 2-anilino-4-(1H-pyrrol-3-yl)pyrimidines were prepared and evaluated for their ability to inhibit cyclin-dependent kinases (CDKs). A number of analogues were found to be potent CDK2 and CDK4 inhibitors and to exhibit anti-proliferative activity against human tumour cell lines. Structure-activity relationships and biochemical characterization are presented.     

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.     

Identification,characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea,Ctenocephalides felis

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.     

Synthesis, characterization and antiamoebic activity of 1-(thiazolo[4,5-b]quinoxaline-2-yl)-3-phenyl-2-pyrazoline derivatives

A new series of 1-N-thiocarboxamide-3-phenyl-2-pyrazolines 1-6 was synthesized by cyclization of different Mannich bases with unsubstituted thiosemicarbazide. The reaction of cyclized pyrazoline derivatives 1-6 with 2,3-dichloroquinoxaline afforded the title compounds 7-12. The structures of the new compounds were confirmed by elemental analyses as well as (1)H, (13)C NMR, IR and electronic spectral data. The HM1:IMSS strain of Entamoeba histolytica parasite was cultured in vitro and the sensitivity of the parasite to the synthesized compounds was evaluated using the microdilution method. Among all the pyrazoline derivatives 1-6, none was found to be a better inhibitor as compared to the reference drug, metronidazole. The quinoxaline derivatives, 9, 11 and 12 were found to be potent inhibitors of E. histolytica.     

Synthesis and in vitro antimycobacterial activity of novel 3-(1H-pyrrol-1-yl)-2-oxazolidinone analogues of PNU-100480

Pursuing our search program for new antitubercular drugs we decided to explore the potentiality of oxazolidinone moiety by synthesizing novel 3-(1H-pyrrol-1-yl)-2-oxazolidinone analogues of PNU-100480. The new derivatives were tested against atypical mycobacteria as well as against drug resistant Mycobacterium tuberculosis and some of them exhibited a fairly good activity against Mycobacterium avium complex (MAC).     

Synthesis and antibacterial activity of a new series of 3-[3-(substituted phenyl)-1-phenyl-1H-pyrazol-5-yl]-2H-chromen-2-one derivatives

A series of 3-[3-(substituted phenyl)-1-phenyl-1H-pyrazol-5-yl]-2H-chromen-2-one (4a–k) were synthesized by reaction of 3-[2,3-dibromo-3-(substituted phenyl)propanoyl]-2H-chromen-2-one (3 a-k) with phenyl hydrazine in presence of triethylamine in absolute ethanol, characterized by spectral data and screened for their in vitro antibacterial activity against gram-positive and gram-negative bacteria. Among the series, compounds 4d, 4h and 4i displayed an encouraging antibacterial activity profile as compared to reference standard drug ciprofloxacin against tested bacterial strains.     

Potent antimicrobial activity of 3-(4,5-diaryl-1H-imidazol-2-yl)-1H-indole derivatives against methicillin-resistant Staphylococcus aureus

A new series of antimicrobial derivatives [3-(4,5-diaryl-1H-imidazol-2-yl)-1H-indole)] have been synthesized with potent activity against strains of Staphylococcus aureus, including methicillin-resistant strains (MRSA). Compound 17 [3-(4,5-bis(4-fluorophenyl)-1H-imidazol-2-yl)-5-bromo-1H-indole], the most active derivative was shown to inhibit the growth of all Gram-positive strains tested, including vancomycin resistant Enterococcus faecalis and Enterococcus faecium with no activity against Gram-negative bacteria.