Epidemiological aspects on vector-borne infections in stray and pet dogs from Romania and Hungary with focus on Babesia spp.

Canine arthropod-borne infections are of major interest in small animal practice and have been widely investigated in Central and Western Europe. However, only limited epidemiological data are available from South-Eastern European countries, although diseases including babesiosis or dirofilariosis are widely recognised as important canine infections in these countries. A steadily increasing number of dogs imported from South-Eastern Europe into Germany require particular attention by small animal practitioners. In this study, a total of 216 dogs [29 local Romanian pet dogs presented at Salvavet Veterinary Clinic in Bucharest, Romania, and 187 imported stray dogs from Romania (n = 109) and Hungary (n = 78) into Germany] were screened by molecular biological, serological and haematological methods for canine arthropod-borne infections. Eleven different parasitic and bacterial vector-borne pathogens-Babesia canis canis, Babesia canis vogeli, Babesia gibsoni, Babesia felis-like, Hepatozoon canis, Leishmania spp., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum, Anaplasma phagocytophilum and Mycoplasma haemocanis-were detected. Fifty-six percent of the dogs were positive by direct methods. B. canis canis was the most prevalent pathogen in dogs imported to Germany (42.8%) and dogs submitted for clinical consultation in Bucharest (44.8%). Our data strongly suggest the introduction of an adjusted screening panel in dogs from South-East Europe in view of increasing importation of dogs into Germany.

Characterization of the allergen filarial tropomyosin with an invertebrate specific monoclonal antibody.

Tropomyosins of invertebrates are pan-allergens responsible for wide spread allergic reactions against seafood and arthropods. As invertebrate tropomyosins are highly conserved, helminth tropomyosins are likely to show properties similar to these medically important allergens. Studies with a monoclonal antibody, NR1, raised against tropomyosin of the rodent filarial nematode Acanthocheilonema viteae revealed a B cell epitope common to helminths and marine mollusks, which does not occur in vertebrate tropomyosin. This antibody detected tropomyosin of A. viteae, other filariids, nematodes, trematodes and a cestode, and recognized as well tropomyosin of oyster, squid and octopus, but not of arthropods and vertebrates. Immunohistological analyses of A. viteae, Onchocerca volvulus and other nematodes using NR1 showed that tropomyosin is located in the fibrillar part of the body wall muscles and the uterus, and is also conspicuous in muscles of the pharynx, the vagina and other organs of the nematodes. The abundance of a pan-allergen like tropomyosin in parasitic worms and the counterintuitive, but well documented protection against allergic reactivity by some chronic helminth infections is discussed.

Modelling and simulating interleukin-10 production and regulation by macrophages after stimulation with an immunomodulator of parasitic nematodes.

Parasitic nematodes can downregulate the immune response of their hosts through the induction of immunoregulatory cytokines such as interleukin-10 (IL-10). To define the underlying mechanisms, we measured in vitro the production of IL-10 in macrophages in response to cystatin from Acanthocheilonema viteae, an immunomodulatory protein of filarial nematodes, and developed mathematical models of IL-10 regulation. IL-10 expression requires stimulation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and p38, and we propose that a negative feedback mechanism, acting at the signalling level, is responsible for transient IL-10 production that can be followed by a sustained plateau. Specifically, a model with negative feedback on the ERK pathway via secreted IL-10 accounts for the experimental data. Accordingly, the model predicts sustained phospho-p38 dynamics, whereas ERK activation changes from transient to sustained when the concentration of immunomodulatory protein of Acanthocheilonema viteae increases. We show that IL-10 can regulate its own production in an autocrine fashion, and that ERK and p38 control IL-10 amplitude, duration and steady state. We also show that p38 affects ERK via secreted IL-10 (autocrine crosstalk). These findings demonstrate how convergent signalling pathways may differentially control kinetic properties of the IL-10 signal.

Chitinase genes expressed by infective larvae of the filarial nematodes, Acanthocheilonema viteae and Onchocerca volvulus.

State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.

Prevalencia de Dirofilaria immitis en perros callejeros de la ciudad de Mérida, Yucatán, México / Prevalence of Dirofilaria immitis in stray dogs in the city of Merida, Yucatan, Mexico

Se estudió la dirofilariasis canina en la ciudad de Mérida Yucatán, en el sureste de México, con el objetivo de determinar su prevalencia y comparar la efectividad de la prueba de gota gruesa y Knott modificada , con la presencia de parásitos adultos en la necropsia. prevaleció Dirofilaria immitis en 6.54 por ciento y Dipetalonema reconditum en 7.47 por ciento. La prueba de Knott modificada detectó 11 casos positivos a microfiliarias circulantes y la prueba de gota gruesa solamente detectó 3 casos. Se encontraron 2 casos de filariasis mixta. Cinco animales fueron positivos a parásitos adultos en la necropsia y 7 positivos a microfiliarias circulantes; sin embargo, 2 casos con parásitos adultos en la necropsia no pudieron ser detectados por la técnica de knott modificada, y 2 casos positivos a Knott modificada no presentaron parásitos adultos a la necropsia. Se presentó un promedio de 6 parásitos adultos por cada positivos; los parásitos fueron localizados en distintas partes del corazón. Se hallaron 3 casos positivos a larvas de Dipetalonema reconditum en la orina, lo que representa 5.54 por ciento de positividad. Se concluye que para el diagnóstico de la dirofilariasis canina es necesario la detección de microfilarias en sangre y acompañarse de pruebas complementarias

External stimuli and intracellular signalling in the modification of the nematode surface during transition to the mammalian host environment.

Previous work has shown that the surface of infective larvae of parasitic nematodes will not bind the fluorescent lipid analogue 5-N-(octadecanoyl)aminofluorescein (AF18) until after exposure of the parasite to mammalian tissue-culture conditions. In this study, culture media which are permissive or non-permissive for the acquisition of lipophilicity for AF18 were altered in order to examine possible stimuli involved. This showed that external alkaline pH and high sodium ion concentration were highly stimulatory. The internal signalling pathways which may be involved in the surface alteration were then examined using agents which are known to affect intracellular signalling in mammalian cells. The results indicated that elevation of cGMP levels was stimulatory whereas inhibition of a putative Na+/H+ antiporter or calcium mobilization was inhibitory, and it is argued that high intracellular levels of cAMP may be inhibitory. Whilst the precise effects of the agents used on nematode cells remain to be established, these results provide a framework for the examination of the processes involved in the modification of the nematode surface which takes place immediately after the infection event.

Recovery, distribution, and development of Acanthocheilonema viteae third- and early fourth-stage larvae in adult jirds.

Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates.

Cryopreservation of infective larvae of Dipetalonema viteae.

infective larvae of Dipetalonema viteae produced infections in Mongolian jirds (Meriones unguiculatus) after storage of infected ticks (Ornithodoros tartakovskyi) in the presence of dimethyl sulfoxide (DMSO, 5%) for 7 or 595 days in liquid nitrogen (-196 C). Infectivity of these larvae was only partially impaired. Microfilaremias of test jirds were generally lower than those of control jirds given nonfrozen larvae; however, the majority of test jirds developed microfilarial counts suitable for use in infecting ticks. In contradistinction, larvae frozen free of the tick failed to retain infectivity. Apparently the tick, in conjunction with DMSO, protects the larvae during freezing and thawing.