Flea species infesting dogs in Spain: updated spatial and seasonal distribution patterns

This entomological survey examines the spatial and seasonal distribution patterns of flea species infesting dogs in Spain. Bioclimatic zones covering broad climate and vegetation ranges were surveyed according to size. In a cross‐sectional spatial survey carried out from late May 2013 to mid‐July 2015, 1084 dogs from 42 different locations were examined. A total of 3032 fleas were collected and identified as belonging to the following species: Ctenocephalides felis (Siphonaptera: Pulicidae) (81.7%, 2476 fleas); Ctenocephalides canis (11.4%, 347 fleas); Pulex irritans (Siphonaptera: Pulicidae) (6.9%, 208 fleas), and Echidnophaga gallinacea (Siphonaptera: Pulicidae) (0.03%, one flea). Variables observed to have effects on flea abundance were animal weight, sex, length of hair and habitat. In the seasonal survey conducted from June 2014 to June 2015, 1014 fleas were collected from 239 dogs at 30 veterinary practices across Spain. Peaks in C. felis abundance were observed in early summer and late autumn, whereas high numbers of P. irritans and C. canis were recorded in autumn. Numbers of fleas detected in winter were low overall. Based on these findings, the present study updates the spatial and seasonal distributions of flea species in Spain and assesses the impacts of host and habitat variables on flea infestation.     

Molecular detection of zoonotic bartonellae (B. henselae,B. elizabethae and B. rochalimae) in fleas collected from dogs in Israel

Fleas represent an acknowledged burden on dogs worldwide. The characterization of flea species infesting kennel dogs from two localities in Israel (Rehovot and Jerusalem) and their molecular screening for Bartonella species (Rhizobiales: Bartonellaceae) was investigated. A total of 355 fleas were collected from 107 dogs. The fleas were morphologically classified and molecularly screened targeting the Bartonella 16S–23S internal transcribed spacer (ITS). Of the 107 dogs examined, 80 (74.8%) were infested with Ctenocephalides canis (Siphonaptera: Pulicidae), 68 (63.6%) with Ctenocephalides felis, 15 (14.0%) with Pulex irritans (Siphonaptera: Pulicidae) and one (0.9%) with Xenopsylla cheopis (Siphonaptera: Pulicidae). Fleas were grouped into 166 pools (one to nine fleas per pool) according to species and host. Thirteen of the 166 flea pools (7.8%) were found to be positive for Bartonella DNA. Detected ITS sequences were 99–100% similar to those of four Bartonella species: Bartonella henselae (six pools); Bartonella elizabethae (five pools); Bartonella rochalimae (one pool), and Bartonella bovis (one pool). The present study indicates the occurrence of a variety of flea species in dogs in Israel; these flea species are, in turn, carriers of several zoonotic Bartonella species. Physicians, veterinarians and public health workers should be aware of the presence of these pathogens in dog fleas in Israel and preventive measures should be implemented.     

Intracellular localization of the hydrogenase in Desulfotomaculum orientis

Abstract Cell extracts of Desulfotomaculum orientis , grown with H₂ plus sulfate as sole energy source, revealed hydrogenase activities between 0.3 and 2 μmol H₂ per min and mg protein when methyl viologen was used as electron acceptor. With benzyl viologen, methylene blue, FAD or FMN, lower activities were found; NAD was not reduced. The hydrogenase activity was strongly inhibited by CuCl₂; however, copper inhibition was not observed with whole cells, indicating that the hydrogenase is located intracellularly. After high-speed centrifugation of cell-free extracts, varying proportions, between 11 and 90%, of the hydrogenase were detected in the soluble fraction, the rest being associated with the membrane fraction.     

Observations on the susceptibility of Ctenocephalides felis (Siphonaptera: Pulicidae) to malathion and permethrin in Tanzania

Laboratory-reared Ctenocephalides felis (Bouche) adults were tested with 0.5% malathion and 0.5% permethrin, using the standard WHO methods. After 24 h exposure to malathion (3.6 mg/cm2), 92% of the fleas died. The LT50 for malathion was approximately 8 h. Permethrin (0.45 mg/cm2) produced 100% mortality of exposed insects after 24 h while with a higher dose (0.9 mg/cm2) all fleas died after 8 h exposure. LT50 for the two doses of permethrin were 7.7 and 1.05 h, respectively. The failure of the diagnostic dose of malathion to kill 100% of the population was attributed to resistance. Permethrin is a suitable pesticide for controlling fleas of domestic animals in Tanzania.     

Large-scale monitoring of imidacloprid susceptibility in the cat flea, Ctenocephalides felis

Although on-animal topical treatment with compounds such as imidacloprid has revolutionized the control of the cat flea, Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae), the development of insecticide resistance is a continuing threat. As part of a highly co-ordinated and unprecedented resistance monitoring programme for C. felis, 1437 flea isolates were collected by veterinary clinics in Australia, Germany, France, the U.K. and 29 states in the U.S.A. from 2002 to 2009. About 65% of the collections were made from June to October each year and 71% of the collections were from cats. Collections of flea eggs were sent to one of five different laboratories, where they were tested with a diagnostic dose of imidacloprid (3 p.p.m.) applied to larval flea-rearing medium. Of the 1437 collections received, 1064 contained adequate numbers of eggs for testing. Of these isolates, untreated eggs failed to hatch in 22.7% and were not considered valid bioassays. Survival rates >5% and development of adult fleas (a threshold for further testing) occurred in only 22 isolates. They were re-tested with the same diagnostic dose and none produced >5% adult emergence. Complete dose-response bioassays were performed on three of the isolates that had triggered a second test and produced slopes, intercepts and LC(50) values similar to those for existing susceptible laboratory strains. Results confirmed sustained susceptibility of C. felis to imidacloprid, despite its widespread use for over a decade.     

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.     

Understanding attraction stimuli of the cat flea, Ctenocephalides felis, in non-chemical control methods

Comparisons were conducted of flea catches of four commercially available flea traps in the laboratory and under field conditions, in both rural and urban locations. The results clearly showed the My Flea Trap?, which utilizes an intermittent light to attract fleas, to be far superior in trapping ability to the three continuous light traps; it caught up to 23 times as many fleas as the other traps. Altering the lighting mechanism to provide continuous rather than intermittent light significantly decreased the number of fleas captured. In addition, the use of a green filter significantly increased trapping efficiency, whereas the addition of a heat source had no apparent effect.     

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.     

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.     

Real‐time and multiplex real‐time polymerase chain reactions for the detection of Bartonella henselae within cat flea,Ctenocephalides felis,samples

Bartonella henselae (Rhizobiales: Bartonellacae), the agent of cat‐scratch disease, is an emerging bacterial pathogen which can be transmitted via infective faecal material of Ctenocephalides felis Bouché (Siphonaptera: Pulicidae). Worldwide, B. henselae has been identified in 1–53% of felines and 2.9–17.4% of fleas. Although culture is the routine method for detection, the procedure is time‐consuming and is rarely used for isolation directly from flea vectors. The current study reports the development of a quantitative real‐time polymerase chain reaction (qPCR) to detect and quantify B. henselae organisms from vector samples. The qPCR is specific and detects as few as 2.5 genome copies. To enable direct quantification of Bartonella organisms in different vector samples, we developed a qPCR to detect C. felis DNA that also acts as an extraction control. Combining both PCRs into a multiplex format validates B. henselae results when sampling flea populations, although there is a reduction in sensitivity. This reduction might be counteracted by a different combination of probe fluorophores.     

Detection of Rickettsia spp. and Bartonella spp. in Ctenocephalides felis fleas from free‐ranging crab‐eating foxes (Cerdocyon thous)

Fleas are insects with a worldwide distribution that have been implicated in the transmission of several pathogens. The present study aimed to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Bartonella spp. (Rhizobiales: Bartonellaceae) in fleas from free‐ranging crab‐eating foxes Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from Rio Grande do Sul, southern Brazil. Fleas were collected manually from animals and used for the molecular detection of Rickettsia spp. and Bartonella spp. Twenty‐nine C. thous were sampled in six municipalities. Four foxes were parasitized by 10 fleas, all of which were identified as Ctenocephalides felis (Bouché, 1935) (Siphonaptera: Pulicidae). DNA from Rickettsia felis Bouyer et al., 2001 and Rickettsia asembonensis Maina et al., 2016 were found in three and eight fleas, respectively. In four fleas, DNA of Bartonella sp. was identified. Phylogenetic analysis grouped Bartonella sp. together with other genotypes previously reported in C. felis worldwide. The scenario described in the present study highlights a Neotropical canid parasitized by the invasive cosmopolitan cat flea, which in turn, is carrying potentially invasive vector‐borne microorganisms. These findings suggest that C. felis is adapted to wild hosts in wilderness areas in southern Brazil, hypothetically exposing the Neotropical fauna to unknown ecological and health disturbances.     

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.     

New records for the bee fauna of Iran (Hymenoptera: Apoidea)

In a survery of the bee fauna of Horand forests in East Azerbaijan province of Iran during 2008 and 2009, 46 species belonging to the families Anthophoridae, Halictidae and Megachilidae were identified. Eight species are new to the fauna of Iran: Evylaeus albipes (Fabricius, 1781), E. corvinus (Morawitz, 1878), E. israelensis (Ebmer, 1974), Seladonia desertorum (Morawitz, 1876), Heriades crenulatus Nylander, 1856, Hoplitis anthocopoides (Schenck, 1853), H. manicata (Morice, 1901) and H. rufohirta (Latreille, 1811).     

Endosymbionts of Ctenocephalides felis felis (Siphonaptera: Pulicidae) obtained from dogs captured in Belo Horizonte, Minas Gerais, Brazil

Specimens of fleas Ctenocephalides felis felis (1052 female symbol/448 male symbol), obtained from 150 dogs in Centro de Controle de Zoonoses de Belo Horizonte, Minas Gerais, Brazil, were dissected and examined for endosymbionts. Three protozoan, Nolleria pulicis, a gregarine (Actinocephalidae) and Leptomonas sp., together with one cestode, Dipylidium caninum were identified. Infections by N. pulicis and Leptomonas sp. occurred mainly in the warm-rainy period. The prevalence and distribution of these endosymbionts in fleas derived from Brazil and South America, and the their variation according to sex and season, are reported for the first time.     

Shark scavenging behavior in the presence of competition

The distribution of organisms within a community can often be determined by the degree of plasticity or degree of specialization of resource acquisition. Resource acquisition is often based on the morphology of an organism, behavior, or a combination of both. Performance tests of feeding can identify the possible interactions that allow one species to better exploit a prey item. Scavenging behaviors in the presence or absence of a competitor were investigated by quantifying prey selection in a trophic generalist, spiny dogfish Squalus acanthias, and a trophic specialist, smooth-hounds Mustelus canis, in order to determine if each shark scavenged according to its jaw morphology. The diet of dogfish consists of small fishes, squid, ctenophores, and bivalves; they are expected to be nonselective predators. Smooth-hounds primarily feed on crustaceans; therefore, they are predicted to select crabs over other prey types. Prey selection was quantified by ranking each prey item according to the order it was consumed. Dietary shifts were analyzed by comparing the percentage of each prey item selected during solitary versus competitive scavenging. When scavenging alone, dogfish prefer herring and squid, which are easily handled by the cutting dentition of dogfish. Dogfish shift their diet to include a greater number of prey types when scavenging with a competitor. Smooth-hounds scavenge on squid, herring, and shrimp when alone, but increase the number of crabs in the diet when scavenging competitively. Competition causes smooth-hounds to scavenge according to their jaw morphology and locomotor abilities, which enables them to feed on a specialized resource [Current Zoology 56 (1): 100-108 2010].     

Comparative efficacy on dogs of a single topical treatment with the pioneer fipronil/(S)-methoprene and an oral treatment with spinosad against Ctenocephalides felis

In the study reported here, the pioneer fipronil/(S)-methoprene topical product (FRONTLINE^® PLUS, Merial Limited, Duluth, GA) was compared to the oral spinosad product (COMFORTIS^® Elanco, Greenfield, IN) for efficacy against adult fleas and preventing egg production. The product presentations, doses and labelling were the one applicable in the USA. Using a standard protocol, 200 cat fleas of mixed sex were applied to dogs on Days 1, 7, 14, 21, 28, 35, and 42. Dogs were combed to remove fleas 24 hours post-infestation, the fleas were counted, collected, and then reapplied to each dog following completion of their respective count. At 48 hours post-infestation, comb counts were performed and fleas were removed. No fleas were collected from any dog in the fipronil/(S)-methoprene group at any 24 or 48 hours post-infestation assessment throughout the six weeks study, yielding a preventive efficacy of 100%. For the spinosad treatment, efficacy was 100% at 24 hours and 48 hours through Day 16, and thereafter declined. The results observed in the spinosad-treated dogs were highly variable between animals. At the 24 and 48 hours counts following the Day 21 infestation, only five of eight spinosad-treated dogs (62.5%) were flea-free. Following the Day 28 infestation, spinosad efficacy fell to 85% and 89%, for the 24 hours and 48 hours counts, and only two dogs (25%) were flea free, compared to 100% flea-free dogs in the fipronil/(S)-methoprene group. No fleas were collected from the fipronil/(S)- methoprene treated dogs throughout the entire study, therefore, no eggs were collected at any time from any dog in the group. However, in the spinosad group adult fleas were found on dogs starting on Day 21 and by Day 30, 42 eggs were collected from one dog that had 107 adult fleas counted at 48 hours. At Day 37 and Day 49, more than 100 eggs were collected from each dog in the spinosad-treated and control groups.     

Evidence for the presence of enkephalins in the heart

Extracts of guinea pig hearts were subjected to high performance liquid chromatography (HPLC) and the eluted fractions monitored by radioimmunoassays (RIA) for their content of leucine⁵-enkephalin (Leu-ENK) and methionine⁵ enkephalin (Met-ENK). Distinct peaks of both Leu-ENK and Met-ENK immunoreactivity were found corresponding to the position of synthetic Leu-ENK and Met-ENK respectively. The ratio of Leu-ENK to Met-ENK content was about 1:4. Chemical sympathectomy with 6-hydroxydopamine (6-OH-DA) produced a dramatic fall in noradrenaline content of the heart by more than 99%, whereas the concentration in Leu-ENK was reduced by only 70%. The Leu-ENK content of the adrenal glands was not affected by this treatment. These observations point to an enkephalinergic innervation of the heart which appears to be mainly of sympathetic origin. The results suggest the participation of enkephalins in cardiac reflex mechanisms.     

Variation in the load of the horn fly, Haematobia irritans, in cattle herds is determined by the presence or absence of individual heifers

The distribution of horn flies, Haematobia irritans (L.) (Diptera: Muscidae), in herds of Danish Holstein-Friesian cattle was investigated in two studies conducted during two field seasons. In the first study, highly significant differences in fly distribution between the most and the least fly-susceptible heifers were observed. In one herd, the mean difference between the most fly-susceptible and the most fly-resistant heifers was 268 Ha. irritans specimens. The highest ratio between upper and lower mean fly number was 64.1:1, whereas the lowest was 3.1:1. In the second year, it was demonstrated that the heifers kept their rank in fly attraction over time. The trial clearly demonstrated that some heifers were attracting flies, whereas others, even in the same herd, only carried a few. In the second study, heifers were moved in and out of herds in an attempt to manipulate fly loads in the herds. In year 1, one herd (herd A) received four fly-resistant heifers from another herd (herd B), resulting in a drop in the mean number of flies, whereas herd B received four fly-susceptible heifers from herd A, resulting in an elevation of the mean number of flies. In year 2, a similar pattern emerged using herds C and D, and when the cattle were later returned to their original herds, the fly loads returned to their original distribution. The data presented here show unequivocally that, for horn flies, there can be considerable differences in fly loads for individual heifers within the Holstein-Friesian breed. Furthermore, the overall fly load within herds can be manipulated, and can be reversed. Thus, the distribution in the number of flies within a herd appears to depend on the number of fly-resistant or fly-susceptible heifers. The possible role of chemical factors emitted by heifers, i.e. volatile semiochemicals, in determining differences in fly loads is discussed, whereby attractants are emitted by fly-susceptible heifers and enable flies to locate their host, and repellents are emitted by fly-resistant heifers such that the flies are actively repelled from the herd.