Concomitant immunity in a rodent model of filariasis: the infection of Meriones unguiculatus with Acanthocheilonema viteae.

In an attempt to study the occurrence of concomitant immunity in filarial infections, jirds (Meriones unguiculatus) were experimentally infected with Acanthocheilonema viteae, and patent animals were superinfected with a defined dose of A. viteae stage 3 larvae (L3). Infected animals harbored significantly less worms deriving from the superinfection than the control group (P < 0.05, 56.2%, and 63.4% protection), as shown by analysis of female worms 6 wk after superinfection on the basis of their developmental status and their length. This protection was not due to contact with L3 antigens because a significant reduction of worm burdens deriving of a superinfection was also observed after subcutaneous implantation of a single female worm (P < 0.05, 40.2% and 64.9% protection). The induced protective responses target L3 and restrict their migration because an established infection resulted in a reduction of L3 recovery (95.6% and 94.3%, P < 0.001) from tissues of jirds at day 5 after superinfection. Other data show that L3 from a superinfection are trapped within eosinophil-rich granulomas, which is likely to create unfavorable conditions for the worms and to lead to later death. Taken together, established A. viteae-infections partially protect hosts against homologous superinfection by an immune-mediated mechanism and, thus, regulate the population density of the parasites within the host by concomitant immunity.

Up-regulation of extracellular copper/zinc superoxide dismutase mRNA after transmission of the filarial parasite Acanthocheilonema viteae in the vertebrate host Meriones unguiculatus.

The gene encoding the cytoplasmic copper/zinc superoxide dismutase (AVSOD1) from the filarial parasite Acanthocheilonema viteae was isolated from a genomic DNA library using a degenerate oligonucleotide probe. Additionally, cDNAs of the AVSOD1 and the secreted extracellular SOD (AVSOD2) were both cloned by RT-PCR, and the AVSOD2 was expressed at high levels in E. coli. The amino acid sequence of the AVSOD1 is 89.5 and 87.5% identical to that of the corresponding enzymes of Brugia pahangi and Onchocerca volvulus, respectively. In contrast, the AVSOD2 shows a lower degree of identity to the other filarial SODs and is extensively glycosylated. RT-PCR studies demonstrate the expression of both SOD subtypes in all developmental stages of A. viteae and indicate up-regulation of the AVSOD2 expression after transmission from the vector to the definitive host. This suggests an enhanced requirement for SOD activity in post-infective larval stages and adults of A. viteae. ELISAs performed with purified recombinant AVSOD2 show that the AVSOD2 is not a major target for the immune system in naturally infected jirds.

Experimental transmission of Dipetalonema dracunculoides (Cobbold 1870) by Rhipicephalus sanguineus (Latreille 1806).

A dog naturally infected with Dipetalonema dracunculoides and having a microfilaremia of 6050 microfilariae per mm3 of blood was used as source of infection. Experimentally cultivated nymphs of Rhipicephalus sanguineus were fed on the donor dog. Once engorged, ninety-three nymphs were removed and kept at 30 degrees C and RH 90% until they moulted to the adult stage. To study the development of microfilariae in the vector, ten ticks were dissected at Day 37 post-infection. Adult infected ticks were fed on two uninfected dogs. The observed pre-patent periods were 69 and 76 days. Trans-stadial transmission of Dipetalonema dracunculoides by Rhipicephalus sanguineus was demonstrated.

Canine filariasis. Importance and transmission in the Baix Llobregat area, Barcelona (Spain).

Field and laboratory studies were performed in order to assess the degree of canine dirofilariasis caused by Dirofilaria immitis (Leidy) in the Baix Llobregat region, a fluvial area near Barcelona, Spain. A total of 188 dogs were sampled between May and August of 1994. Three main areas were chosen: the Western Delta, the Eastern Delta and the Northern zone. Simultaneously, a mosquito sampling programme was carried out with CO2 light traps, to search for infective larvae (L3) of D. immitis. Of the 188 dogs sampled, 38 were positive for at least one of the three filaria found: D. immitis 12.8%, Dipetalonema reconditum (Grassi) 3.7% and Dipetalonema dracunculoides (Cobbold) 2.7%. Only 1.1% showed a mixed infection of both D. immitis and D. dracunculoides. Although Dirofilaria repens Raillet et Henry has been found in Spain, it was not found in this study. Comparing the three zones of the Baix Llobregat, the Eastern Delta showed the highest level of D. immitis (35.3%), probably due to the presence of Aedes caspius (Pallas). Despite the effort in sampling the mosquito population, D. immitis was not found in any of the 2001 females dissected, belonging to 5 species.

Hippobosca longipennis--a potential intermediate host of a species of Acanthocheilonema in dogs in northern India.

BACKGROUND: Hippobosca longipennis (the 'dog louse fly') is a blood sucking ectoparasite found on wild carnivores such as cheetahs and lions and domesticated and feral dogs in Africa, the Middle East and Asia, including China. Known as an intermediate host for Acanthocheilonema dracunculoides and a transport host for Cheyletiella yasguri, it has also been suggested that H. longipennis may be a vector for other pathogens, including Acanthocheilonema sp.? nov., which was recently reported to infect up to 48% of dogs in northern India where this species of fly is known to commonly infest dogs. To test this hypothesis, hippoboscid flies feeding on dogs in Ladakh in northern India were collected and subjected to microscopic dissection. RESULTS: A total of 12 infective larvae were found in 10 out of 65 flies dissected; 9 from the head, 2 from the thorax and 1 from the abdomen. The larvae averaged 2, 900 (± 60) µm in length and 34 (± 5) µm in width and possessed morphological features characteristic of the family Onchocercidae. Genetic analysis and comparison of the 18S, ITS-2, 12S and cox-1 genes confirmed the identity of the larvae as the Acanthocheilonema sp.? nov. reported in dogs in Ladakh. CONCLUSION: This study provides evidence for a potential intermediate host-parasite relationship between H. longipennis and the canine Acanthocheilonema sp.? nov. in northern India.

First report of Acanthocheilonema spirocauda in the Mediterranean monk seal (Monachus monachus).

The Mediterranean monk seal (Monachus monachus) is one of the world's most endangered marine mammals. The largest population is located mainly throughout the Aegean and Ionian islands and along the coastline of southern continental Greece. We report the findings of a necropsy and discuss their potential importance to the conservation of the species. The adult female monk seal appeared to be in a good nutritional state. The main necropsy findings were injuries consistent with a violent and sudden death, including three round wounds on the ventral surface of the body and several hematomas, as well as a decomposing male fetus in the uterus. Two nematodes were found in the right ventricle of the heart; no abnormalities were observed in the lungs, pulmonary vessels, or heart. The nematodes were identified as Acanthocheilonema spirocauda. This is the first report of infection of the Mediterranean monk seal with A. spirocauda, even though this is the most common heartworm found in most pinnipeds worldwide. This parasite should be considered in health care monitoring projects of this endangered species.

[Experimental infection of soft ticks with Dipetalonema witei (author's transl)]. / Experimentelle Infektion von Lederzecken mit Dipetalonema witei.

The intermediate host of Dipetalonema witei, the soft tick Ornithodoros moubata, can be infected experimentally with microfilariae. Microfilariae isolated from the blood of infected donor animals were injected into the dorsal haemocoel of the ticks where they developed to infectious filariae within approx. 50 days. The rate of infection of ticks was 100% when more than 100 microfilariae were injected. An average of 23% was recovered as infectious filariae. Up to 450 ticks can be infected with 1000 microfilariae each from a single donor animal within one year, whereas donor animals can only be used for about 1 month if ticks are naturally infected. The method described has advantages compared to natural tick infections with respect to the rate of infection of ticks, the development of microfilariae, and the amount of infectious material produced.

[Intake of microfilariae by the vector in the case of a low microfilareamia (author's transl)]. / Ingestion des microfilaires par le vecteur dans le cas des basses microfilaremies.

The study of the intake of microfilariae of Dipetalonema dessetae by Aedes aegypti leads to two conclusions with respect to the microfilaraemia: --there is no concentration of microfilariae in the uptake of blood by Aedes. --the microfilariae are nearly homogeneously distributed in the vertebrate host blood available to the vector for feeding. Such a distribution of microfilariae in the cutaneous blood supply of the host gives the maximum chance for a mosquito to become infected when taking a blood meal. In the pair Wuchereria bancrofti--Aedes polynesiensis the capacity of individuals with a low microfilaraemia to transmit infections seems to be related to a limitation phenomenon.

Artificial feeding and successful reproduction in Ornithodoros moubata moubata (Murray, 1877) (Acarina: Argasidae).

The increasing demand for laboratory-reared argasid and ixodid ticks for research and control purposes makes it necessary to develop effective and standardized tick feeding methods without using live animals as hosts. The in vitro maintenance technique, described in this paper, has been used successfully for rearing Ornithodoros moubata moubata by feeding all nymphal and adult instars through Parafilm 'M' sealing film on heparinized bovine blood (fresh standard). The technique is based on a specially designed tick feeding apparatus with a capacity to feed 2000 first nymphal instars (N 1) or up to 200 adults at one time. For different 1-month-old instars feeding rates were between 80-100%. Using this feeding technique the subsequent egg production of female ticks was remarkably high, producing an average of 210.2 eggs per tick with a hatch rate of 96.72%. There was no overall difference in the reproductive capacity of 1-month-old female O. M. moubata fed on heparinized and haemolyzed bovine blood (kept deep-frozen), heparinized rat blood and defibrinated ovine blood when compared with those fed on heparinized bovine blood (fresh standard).

Prenatal sensitisation in experimental filariasis: observations on Acanthocheilonema viteae infections in mice.

The probability that in utero exposure to filarial antigens may influence the outcome of a subsequent infection has been investigated using a laboratory model whereby BALB/c mice are implanted with adult, female Acanthocheilonema viteae in order to generate a high-level, long lasting microfilaraemia. When infected using this procedure, BALB/c and (BALB/c x B10) F1 mice can be defined as susceptible and resistant respectively in terms of the microfilaraemia produced. By using microfilaraemic BALB/c mice as mothers, BALB/c and F1 offspring were exposed to the possibility of in utero infection. The finding of microfilariae in foetal tissues and their presence in the blood of two week old mice confirmed the transplacental transmission of parasites in both cases, BALB/c and F1 progeny born to microfilaraemic mothers failed to support a full infection from the L3 stage; similarly, progeny implanted with female worms were as sus-ceptible and resistant respectively as unexposed BALB/c and F1 controls. Spleen cells from in utero exposed, two week old BALB/c and F1 mice recognised filarial antigens in lymphocyte proliferation assays, as did their microfilaraemic mothers. In Western Blot studies, sera from such mice and from foetal, in utero exposed BALB/c mice recognised the same spectrum of A. viteae antigens as their mothers, which strongly suggests the transplacental transfer of maternal antibody. These results demonstrate that the A. viteae-mouse model may be useful in studying transplacental transmission and prenatal sensitisation in experimental filariasis.

[Imported parasites in dogs: Dirofilaria repens and Dipetalonema reconditum]. / Eingeschleppte Parasiten bei Hunden: Dirofilaria repens und Dipetalonema reconditum.

Between June 1993 and May 1996 infections with filariae were diagnosed in 80 dogs. Five animals were infected with Dirofilaria repens and three other dogs with Dipetalonema reconditum. One of the D. reconditum positive dogs was also infected with Dirofilaria immitis. An epidemiological analysis of the cases was possible for three D. repens and two D. reconditum infected dogs. Two of the dogs with D. repens infections had been imported from Italy or Greece, the third dog had been taken by its owner to Hungary and former Yugoslavia. The dogs infected with D. reconditum were imported from Corse or Spain. Regarding the other 72 dogs with filariae, 45 animals were infected with D. immitis. For 27 dogs being negative in the D. immitis ELISA, microfilariae have been demonstrated. However, a histochemical species differentiation of the microfilariae was not possible because fresh blood smears were not available.

Dipetalonema viteae and Brugia pahangi transplant infections in gerbils for use in antifilarial screening.

Transplanted infections of Dipetalonema viteae and Brugia pahangi have been evaluated as tools for experimental chemotherapy. Attempts were made to establish these filariae in similar pharmacokinetic sites within the same host, so that direct comparisons of in vivo drug susceptibilities could be made. Unfortunately, it was not possible to establish B. pahangi in the subcutaneous tissues, the preferred site of D. viteae. Therefore, intraperitoneal B. pahangi and subcutaneously implanted D. viteae in gerbils were used for the study. D. viteae infections were significantly enhanced by concomitant infections with B. pahangi, while B. pahangi infection rates were unaffected by the presence of D. viteae. Experiments with amoscanate, CGP6140 and Mel W demonstrated the importance of employing both B. pahangi and D. viteae for antifilarial discovery work and the fundamental effect of parasite location on drug efficacy. D. viteae rapidly migrate from the peritoneal cavity of gerbils following implantation; twenty one hours after infection 73% of transplanted worms were found in the subcutaneous tissues. It was shown that the migration response could be used as a stringent parameter for demonstrating antifilarial activity. D. viteae were exposed to antifilarial drugs for 24 hours in vitro, washed and implanted into the peritoneal cavity of gerbils. At autopsy, 5 days later, 10(-8)M ivermectin and milbemycin D had prevented migration; CGP6140, amoscanate, suramin, flubendazole and furapyrimidone were also detected at less than 10(-6)M using this parameter. In all cases the migration response was more sensitive to drugs than parasite kill. Ivermectin's ability to inhibit worm migration through the tissues is discussed, with respect to the role of itinerant males in the reproductive cycle of Onchocerca volvulus.

Pulícidos: aspectos adaptativos y biológicos / Pulicides: adaptative and biological aspects

Las pulgas son insectos hematófagos que se caracterizan por una baja especificidad parasitaria. Son ectoparásitos de distintos animales y del hombre. El cuerpo de estos insectos está comprimido lateralmente y las antenas pueden replegarse dentro de un surco a los costados de la cabeza, características óptimas para un mejor desplazamiento entre los pelos y las plumas de los hospedadores. Los tres pares de patas son fuertes y poderosas, armadas con robustas espinas y en sus extremos con un par de grandes uñas en forma de garfios. El tercer par es el que está adaptado para saltar. Las piezas bucales son sucsopicadoras y modificadas en forma de estiletes. Algunas especies tienen unas hileras de cerdas (ctenidios) en la zona genal y/o en el dorso del primer segmento toráxico que evitan los movimientos de retroceso y la caída del cuerpo del hospedador. Esta característica también dificulta la extracción de los parásitos. La importancia de los pulícidos radica entre otras, por ser transmisores de la peste y el tifus. Se muestran bajo SEM y con microfotografías las adaptaciones al parasitismo, la biología y las afecciones cutáneas que se producen

Pulícidos: aspectos adaptativos y biológicos / Pulicides: adaptative and biological aspects

Las pulgas son insectos hematófagos que se caracterizan por una baja especificidad parasitaria. Son ectoparásitos de distintos animales y del hombre. El cuerpo de estos insectos está comprimido lateralmente y las antenas pueden replegarse dentro de un surco a los costados de la cabeza, características óptimas para un mejor desplazamiento entre los pelos y las plumas de los hospedadores. Los tres pares de patas son fuertes y poderosas, armadas con robustas espinas y en sus extremos con un par de grandes uñas en forma de garfios. El tercer par es el que está adaptado para saltar. Las piezas bucales son sucsopicadoras y modificadas en forma de estiletes. Algunas especies tienen unas hileras de cerdas (ctenidios) en la zona genal y/o en el dorso del primer segmento toráxico que evitan los movimientos de retroceso y la caída del cuerpo del hospedador. Esta característica también dificulta la extracción de los parásitos. La importancia de los pulícidos radica entre otras, por ser transmisores de la peste y el tifus. Se muestran bajo SEM y con microfotografías las adaptaciones al parasitismo, la biología y las afecciones cutáneas que se producen (AU)