High Rickettsial Diversity in Rodents and Their Ectoparasites From the Central Highlands of Madagascar

Rickettsioses are among emerging infectious diseases around the world. In Madagascar, little information is available regarding Rickettsia (Rickettsiales: Rickettsiaceae) diversity and their potential impacts on public health. In fact, molecular screening of ectoparasites of mammals reported the presence of three species, Rickettsia africae, Rickettsia typhi, and Rickettsia felis. The present study aims to investigate the diversity of Rickettsia in small mammals and associated ectoparasites (fleas and ticks) using a molecular approach. In September and December 2016, fieldworks were undertaken in two districts of Madagascar to capture small mammals using standard traps (Tomahawk and Sherman traps) and collect associated ectoparasites. In total, 12 taxa of ectoparasites (5 flea and 7 tick species) were collected from 89 individuals of four species of terrestrial small mammals. Rickettsia spp. were molecularly identified in one specimen of Rattus rattus (Rodentia: Muridae), one specimen of Pulex irritans (Siphonaptera: Pulicidae) as well as four specimens of Ixodes cf. colasbelcouri (Ixodida: Ixodidae). This study showed the presence of three phylogenetically distinct taxa of Rickettsia in small mammals and their ectoparasites. The current study broadens our knowledge on the diversity of Rickettsia in the Central Highlands of Madagascar and highlights for the first time the presence of Ri. felis in R. rattus and in tick, I. cf. colasbelcouri in Madagascar. Additional studies are needed to have exhaustive information on Rickettsia in small mammals and their ectoparasites, to determine their pathogenicity as well as their potential effects on public health in order to update the national policy for the control of emerging infectious diseases in Madagascar.

The comparative evaluation of complex drugs based on fipronil for ectoparasitosis of dogs and cats

The article presents data on the effectiveness of new domestic antiparasitic drug “Insectostop for dogs and cats” in comparison to the reference drugs “Burdi Fipro for dogs” and “Burdi Fipro for cats” that is used for therapeutic and prophylactic purposes against ectoparasitosis of dogs and cats of different breeds. According to the results of the research, the parasitism of fleas of Ctenocephalus canis, C. felis and Pulex irritans species and parasitiform mites of the Ixodes ricinus and Dermacentor reticulatus species and sarcoptiform mites of the species Otodectes cynotis were detected on experimental animals. Clinically, this was inspected by redness, inflammation of the skin, itching reflex, the emergence of papules on the skin, scales. Based on the results, it was found that 8 hours after usage of the experimental drug “Insectostop for dogs and cats” its effectiveness in syphonapterosis of dogs was 87.9 %, and after usage of the reference drug “Bourdie Fipro for dogs” – 86.9 %, and in experiments on cats, the effectiveness of the experimental drug for siphonapterosis in cats was 92.4 %, and the reference drug “Bourdie Fipro for cats” – 90.3 %. Starting from the first and third days after usage of drugs on the fur of dogs and cats of the experimental and control groups, parasitological studies did not reveal fleas of the species Ctenocephalus canis, C. felis and Pulex irritans. So starting from the first day, the experimental and reference drugs showed 100 % effectiveness during the siphonapterosis in dogs and cats. During the ixodidosis of dogs, it was found that at the 8-th hour after usage of the experimental drug “Insectostop for dogs and cats” its effectiveness was 82.9 %, and after usage of the reference drug “Bourdie Fipro for dogs” – 81.3 %. At the 24-th hour after usage of the experimental drug, its effectiveness was 97.6 %, and after usage of the reference drug – 95.9 %. At the 72nd hour of the experiment, no adult representatives of the species Ixodes ricinus and Dermacentor reticulatus were found on the fur of dogs of these groups. Thus, at 72 hours after usage of the experimental and reference drug, they showed 100 % efficiency on adult Ixodes mites. During the Otodectesis of dogs it was found that on the 7th day, effectiveness of the experimental drug was 75.6 %, on the 14th day – 98.1 % and after usage of the reference drug “Bourdi Fipro for dogs” its effectiveness on the 7th day was 73.5 %, and on the 14th – 98.3 %. At 21-st days after treatment of animals with drugs as a result of clinical examination and parasitological examination of dogs mites of the species Otodectes cynotis were not detected. In experiments on cats, on the 7th day after usage of experimental drug, its effectiveness during Otodectesis of cats was 76.9 %, and after usage of reference drug – 77.1 %. As a result of clinical examination and parasitological study of the experimental and control groups of animals on the 14th day after treatment of the auricles of cats with mites of the species Otodectes cynotis was not detected. Thus, usage of both drugs promotes the release of Otodectesis in dogs from parasites on the 21st day of the experiment, and cats – on the 14th day. Tests have shown that the experimental drug “Insectostop for dogs and cats” (100 ml of the drug contains the active substance: fipronil – 10 g) does not cause skin irritation, dermatitis, seborrhea, allergic and other side effects, that the drug is well tolerated by dogs and cats and doesn’t give any side effects and changes of clinical condition of animals.

Poor vector competence of the human flea, Pulex irritans, to transmit Yersinia pestis

Background The human flea, Pulex irritans, is widespread globally and has a long association with humans, one of its principal hosts. Its role in plague transmission is still under discussion, although its high prevalence in plague-endemic regions and the presence of infected fleas of this species during plague outbreaks has led to proposals that it has been a significant vector in human-to-human transmission in some historical and present-day epidemiologic situations. However, based on a limited number of studies, P. irritans is considered to be a poor vector and receives very little attention from public health policymakers. In this study we examined the vector competence of P. irritans collected from foxes and owls in the western United States, using a standard protocol and artificial infection system. Methods Wild-caught fleas were maintained in the laboratory and infected by allowing them to feed on human or rat blood containing 2 × 108 to 1 × 109Y. pestis/ml. The fleas were then monitored periodically for infection rate and bacterial load, mortality, feeding rate, bacterial biofilm formation in the foregut (proventricular blockage), and ability to transmit Y. pestis after their single infectious blood meal. Results P. irritans were susceptible to infection, with more than 30% maintaining high bacterial loads for up to 20 days. Transmission during this time was infrequent and inefficient, however. Consistent with previous studies, a low level of early-phase transmission (3 days after the infectious blood meal) was detected in some trials. Transmission at later time points was also sporadic, and the incidence of proventricular blockage, required for this mode of transmission, was low in fleas infected using rat blood and never occurred in fleas infected using human blood. The highest level of blockage and transmission was seen in fleas infected using rat blood and allowed to feed intermittently rather than daily, indicating that host blood and feeding frequency influence vector competence. Conclusions Our results affirm the reputation of P. irritans as a feeble vector compared to rodent flea species examined similarly, and its vector competence may be lower when infected by feeding on bacteremic human blood. Graphic abstract

Field study to investigate the effectiveness and safety of a novel orally administered combination drug product containing milbemycin oxime and lotilaner (Credelio® Plus) against natural flea and tick infestations on dogs presented as veterinary patients in Europe

Background A pivotal randomised, blinded, positive-controlled, multicentre, European field study was conducted to evaluate the effectiveness and safety of a novel combination tablet of lotilaner and milbemycin oxime (Credelio® Plus) administered orally to client-owned dogs naturally infested with fleas and/or ticks. Methods In this field study, households with flea- or tick-infested dog(s) were enrolled on Day 0 into the study to provide data for either the tick or flea infestation cohorts. Households were randomised in a 2:1 ratio to receive either the combination investigational product (IP, Credelio Plus® tablets) or the control product (CP: Nexgard Spectra® tablets). Dogs were administered IP (flea cohort n = 135; tick cohort: n = 147) or CP (flea cohort: n = 67; tick cohort: n = 74) once every 4 weeks for a total of three times at a dose rate of 20.0–41.5 mg/kg bodyweight lotilaner and 0.75–1.53 mg/kg bodyweight milbemycin oxime (IP) or as recommended (CP). Percentage reduction was calculated by comparing individual dog flea and tick counts at each assessed post-treatment time point to their respective baseline (pre-treatment) infestation. Resolution of the clinical signs of flea allergy dermatitis (FAD) was assessed in flea-allergic dogs on the days that flea counts were performed. Results Flea effectiveness of Credelio Plus® after 3 consecutive monthly treatments was 100% against Ctenocephalides felis, C. canis and Pulex irritans. Tick effectiveness of Credelio Plus® over the same time frame was 99.3% for Ixodes ricinus and 100% against Rhipicephalus sanguineus (s.l.). Flea effectiveness of the CP after three consecutive monthly treatments was 100% against C. felis, C. canis and P. irritans. Tick effectiveness of the CP over the same time frame was 99.8% for I. ricinus and 100% against R. sanguineus. Credelio Plus® was well tolerated based on the safety assessments in all treated dogs in this field study. Within both treatment groups there was a reduction in total FAD scores from baseline. Conclusions This pivotal European field study demonstrated the excellent effectiveness and safety of a combination of lotilaner and milbemycin oxime (Credelio Plus®) administered orally to dogs naturally infested with fleas and/or ticks. Graphic Abstract

Διερεύνηση των παθογόνων Bartonella και Rickettsia σε διαβιβαστές και τον άνθρωπο στην Αττική

Η παρούσα διατριβή επικεντρώθηκε στην περιοχή της Αττικής, εστιάζοντας στη μοριακή διερεύνηση κοινών ψύλλων ζώων συντροφιάς για παθογόνα βακτήρια και στην εκτίμηση της φορείας IgG αντισωμάτων στον πληθυσμό έναντι των Rickettsia typhi, Bartonella henselae και B. quintana. Τα δείγματα ψύλλων προήλθαν από κτηνιατρικές κλινικές και τα δείγματα ανθρώπων από ιδιωτικά μικροβιολογικά εργαστήρια στην Αττική. Για την μοριακή διερεύνηση των ψύλλων χρησιμοποιήθηκαν συμβατικές και real-time PCR και αλληλούχιση επόμενης γενεάς του γονιδίου 16S rDNA. Οι ορολογικές δοκιμές έγιναν με έμμεσο ανοσοφθορισμό (Vircell™, Santa Fé, Spain), σύμφωνα με τις οδηγίες του κατασκευαστή. Ο χαρακτηρισμός των βακτηρίων με δυνητική υγειονομική σημασία βασίστηκε στο International Statistical Classification of Diseases and Related Health Problems by WHO (ICD-11, 2019 version).Από τα δείγματα ψύλλων, 100 αποτελούμενα μόνο από θηλυκούς ψύλλους επιλέχθηκαν για ανάλυση, στα οποία περιλαμβάνονταν 96 δείγματα με Ctenocephalides felis, δύο δείγματα με Ctenocephalides canis και δύο δείγματα με Pulex irritans, που συλλέχθηκαν από 67 γάτες και 33 σκύλους.Επιβεβαιωμένα για το γένος Rickettsia βρέθηκαν 16 δείγματα εκ των οποίων 14 επιβεβαιώθηκαν για R. felis εκ των οποίων οκτώ ανήκαν σε γάτα και έξι σε σκύλο. Bartonella spp. ανιχνεύθηκε σε 32 δείγματα. Από αυτά σε 27 ανιχνεύθηκε το είδος: 12 είχαν B. clarridgeiae, επτά B. henselae, ένα B. koehlerae ενώ τα υπόλοιπα επτά είχαν μικτή μόλυνση με περισσότερα από ένα είδη Bartonella. Όλα τα θετικά δείγματα προήλθαν από γάτα εκτός από ένα στο οποίο δεν ταυτοποιήθηκε το είδος. Η ευαισθησία των μοριακών μεθόδων για το γένος Rickettsia κυμάνθηκε από 62,5% έως 93,8% και η ειδικότητα από 65,0% έως 100%. Οι ειδικές για R. felis μοριακές δοκιμές εμφάνισαν 92,9% έως 100% ευαισθησία ενώ η ειδικότητα κυμάνθηκε από 65,0% έως 100%. Δυνητική υγειονομική σημασία στο μικροβίωμα των ψύλλων είχαν 33 γένη βακτηρίων και συγκεκριμένα τα Acinetobacter, Actinomyces, Bacillus, Bacteroides, Bartonella, Bifidobacterium, Brucella, Campylobacter, Clostridium, Corynebacterium, Coxiella, Enterococcus, Fusobacterium, Haemophilus, Kingella, Klebsiella, Legionella, Leptotrichia, Moraxella, Morganella, Mycobacterium, Neisseria, Nocardia, Pasteurella, Propionibacterium, Proteus, Pseudomonas, Rickettsia, Salmonella, Serratia, Staphylococcus, Stenotrophomonas και Streptococcus. Η κατανομή αυτών των γενών δεν διέφερε ανά είδος ζώου ξενιστή παρά μόνο στην Bartonella spp. η οποία ανευρέθηκε συχνότερα σε ψύλλους από γάτες (OR=27,6, p<0,001). Τα γένη Coxiella, Campylobacter, Legionella, Spirochaeta, Salmonella, Brucella ανευρέθησαν μόνο σε γάτες χωρίς όμως να στοιχειοθετείται στατιστική σημαντικότητα. Τα βακτηριακά είδη υγειονομικής σημασίας ήταν 40 και περιλάμβαναν τα εξής: Acinetobacter baumannii, Bacteroides fragilis, Clostridium perfringens, Enterococcus faecalis, E. mundtii, Fusobacterium nucleatum, Haemophilus aegyptius, Kingella kingae, Klebsiella pneumoniae, Leptotrichia buccalis, L. hofstadii, Moraxella lacunata, Pasteurella multocida, Propionibacterium acnes, P. propionicum, Proteus mirabilis, Pseudomonas aeruginosa, Rickettsia australis, R. hoogstraalii, Salmonella enterica, και διάφορα είδη Bartonella, Staphylococcus και Streptococcus. IgG αντισώματα για B. henselae ανιχνεύθηκαν σε 14,3% και για R. typhi σε 1,0% ενώ IgG και για τα δύο παθογόνα σε 3,5% των συμμετεχόντων (Ν=202). Εντοπίστηκε θετική συσχέτιση οροθετικότητας για IgG έναντι B. henselae με κατοίκηση σε παραλιμάνια περιοχή (X2(6)=17,737, p=0,009).Από τα ευρήματα της διατριβής προκύπτει ότι οι ψύλλοι των ζώων συντροφιάς στην Αττική φέρουν σε σημαντικό ποσοστό παθογόνα βακτήρια ενώ οι ψύλλοι από γάτες είναι συχνότερα μολυσμένοι με Bartonella. Η πληθώρα βακτηρίων υγειονομικής σημασίας στους ψύλλους και η αυξημένη και πολύπτυχη διεπαφή του εντόμου αυτού με τον άνθρωπο, ωθούν σε εκτενέστερη διερεύνηση του το κατά πόσο και πως ο ψύλλος σχετίζεται με τη μετάδοση λοιμωδών παραγόντων στον άνθρωπο πέρα από τα γνωστά παθογόνα. Ο πληθυσμός της Αττικής εμφανίζει σε ένα ποσοστό έκθεση σε Bartonella και Rickettsia ενώ ιδιαίτερα για το βακτήριο της Bartonella η πιθανότητα έκθεσης είναι υψηλότερη στην περιοχή του μεγαλύτερου εμπορικού/επιβατικού λιμανιού της χώρας. Συνεπώς, θα πρέπει να ενισχυθεί η επιδημιολογική επιτήρηση και η εργαστηριακή δυνατότητα διάγνωσης για λοιμώξεις οφειλόμενες σε Rickettsia και Bartonella. Παράλληλα θα πρέπει να δοθεί ιδιαίτερη προσοχή σε βλάβες στο καρδιαγγειακό σύστημα που προκαλεί το βακτήριο της Bartonella σε βάθος χρόνου.

An inventory of the types of rodent spread in Houses with a study of external parasites of the most dominant species at Esna area, Luxor Governorate, Egypt

This study was conducted to identify of rodent species and the ectoparasites are widely recognized for the type prevalent in homes, at Esna District, Luxor Governorate, Egypt, during 2020 year. The results were revealed that the presence of three species of rats included gray-bellied rat, Rattus rattus alexandrinus the dominant species from, Rattus rattus frugivorus and the Nile grass rat, Arvicanthis niloticus. The results also indicated that the identification of two types of fleas, Xenopsylla cheopis & Pulex irritans, and one species of lice, Polyplax spinulosa, associated with the gray-bellied was identified. The study reports the interest in making integrated control programs for rodents to get rid of them and the risks of their external parasites. Keywords: Rattus r. alexandrinus, Rattus r. frugivorus, A. niloticus Xenopsylla cheopis,Pulex irritans, Polyplax spinulosa

THE COMPARATIVE EVALUATION OF COMPLEX MEDICINES, BASED ON IMIDACLOPRIDE IN ECTOPARASITOSIS OF DOGS AND CATS

The article represents data on the effectiveness of the new domestic anti-parasitic medicine «Animal VetLine antiparasitic drops for dogs and cats spot-on» in comparison to the reference-medicine «Advantage@» when used for the therapeutic and prophylactic purposes for ectoparasites of dogs and cats of different breeds. According to the results of clinical examination and parasitological examination of animals, parasitism of fleas of the Ctenocephalides canis, Ctenocephalides felis, Pulex irritans species at the intensity of invasion (II) from 5 to 8 specimens/10 cm2, lice Linognathus setosus at II from 1 to 3 specimens/10 cm2 of body surface, and parasitic mites of the Ixodes ricinus, Dermacentor reticulatus species at II from 8 to 12 specimens per animal in addition in dogs. Based on the obtained results for the study of the effectiveness of the «Animal VetLine antiparasitic drops for dogs and cats spot-on» in comparison with the reference drug «Advantage@» medicine, during the infection of dogs and cats by Ctenocephalides canis, Pulex irritans and Ctenocephalides felis fleas, Linognathus setosus lice, mites of the Ixodes ricinus, Dermacentor reticulatus species, was found that on the second day of treatment in the experimental and control groups of animals there was a sharp increase in the number of dead insects, ectoparasites and Ixodes mites, and on the fifth day of the study no live fleas were detected, lice and ticks. At the same time, on the second day after the usage of the experimental and reference drug, their extensile effectivity during the linognatosis of dogs and cats and syphonapterosis of cats was 100.0 %. In the experimental and control groups of dogs on the second day after usage of the experimental drug and the reference drug, the average intensity of invasion of Ctenocephalides canis and Pulex irritans decreased to 1.2±0.4 ectoparasite insects per 10 cm2 of body surface in the experimental group and to 1.4±0.4 insect ectoparasites per 10 cm2 of body surface in the control group of animals, ie the intensity of the experimental drug was 82.1 %, and the reference drug 78.5 %. The results show that the rate of intensification of the drug for exodidosis of dogs in the experimental group on the second day was – 75.0 %, and the control – 68.0 %. However, the average intensity of mite infestation of the family Ixodidae was in the experimental group 2.5±0.15 specimens per animal, and in the control group 3.0±0.21 specimens per animal, respectively. It was found that the experimental drug had no toxic effects and was well tolerated by animals no changes in clinical signs during use were detected.

Presence of the Oriental Rat Flea (Siphonaptera: Pulicidae) Infesting an Endemic Mammal and Confirmed Plague Circulation in a Forest Area of Madagascar

The Oriental rat flea, Xenopsylla cheopis (Rothschild 1903), is a cosmopolitan flea usually found infesting domestic rats. This flea is a well-known major human plague vector in Madagascar. As part of field sampling, fleas and small mammals were collected in the village of South Andranofeno and the natural reserve of Sohisika, two sites of the district of Ankazobe, located in the Central Highlands of Madagascar. Rats inside houses and forest small mammals were trapped using Besancon Technical Services and pitfall traps, respectively. Their fleas were collected and preserved for laboratory works. Collected fleas from the village and forest belonged to five species, which were X. cheopis, Synopsyllus fonquerniei (Wagner and Roubaud 1932) (Siphonaptera: Pulicidae), Echidnophaga gallinacea (Westwood 1875) (Siphonaptera: Pulicidae), Ctenocephalides felisstrongylus (Jordan 1925) (Siphonaptera: Pulicidae), Pulex irritans (Linnaeus 1758) (Siphonaptera: Pulicidae). After sampling in the forest zone, one specimen of X. cheopis was unexpectedly collected while infesting an endemic tenrec Setifer setosus (Schreber 1777) (Afrosoricida: Tenrecidae). Polymerase chain reaction (PCR) diagnosis on all collected fleas allowed detecting plague bacterium Yersinia pestis (Lehmann and Neumann 1896) (Enterobacterales: Yersiniaceae) on nine specimens of the endemic flea S. fonquerniei collected inside forest. The presence of the oriental rat flea in forest highlights the connection between human and wild environments due to animal movements and the fact that the rat flea can infest various hosts. As only one specimen of X. cheopis was collected on S. setosus, we hypothesize that flea was carried from the village to forest. Yersinia pestis infection of forest fleas outlines plague circulation in this sylvatic area.