Rafael Antonio Nascimento Ramos

Development of the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior in Helix aspersa snails.

Aelurostrongylus abstrusus (Strongylida, Angiostrongylidae) and Troglostrongylus brevior (Strongylida, Crenosomatidae) are regarded as important lungworm species of domestic felids, with the latter considered an emerging threat in the Mediterranean region. The present study aimed to assess their concurrent development in the mollusc Helix aspersa (Pulmonata, Helicidae). Thirty snails were infested with 100 first-stage larvae (L1) of A. abstrusus and T. brevior, isolated from a naturally infested kitten. Larval development was checked by digesting five specimens at 2, 6 and 11 days post infestation. Larvae retrieved were morphologically described and their identification was confirmed by specific PCR and sequencing. All H. aspersa snails were positive for A. abstrusus and T. brevior, whose larval stages were simultaneously detected at each time point. In addition, snails were exposed to outdoor conditions and examined after overwintering, testing positive up to 120 days post infestation. Data herein presented suggest that A. abstrusus and T. brevior develop in H. aspersa snails and may eventually co-infest cats. Data on the morphology of both parasitic species in H. aspersa provide additional information on their development and identification, to better understand the population dynamics of these lungworms in receptive snails and paratenic hosts.

Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR.

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult to be differentiated due to the morphological similarities of their first-stage larvae (L1). This paper describes a molecular tool, based on single-step duplex polymerase chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220bp; T. brevior: 370bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ≈ 4.0 to 4.0 × 10(-5) μg/μl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2 × 10(-3) μg/μl of DNA for A. abstrusus, 4.9 × 10(-3)μg/μl for T. brevior, and as low as 4.0 × 10(-3) μg/μl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.

Release of Lungworm Larvae from Snails in the Environment: Potential for Alternative Transmission Pathways

Background Gastropod-borne parasites may cause debilitating clinical conditions in animals and humans following the consumption of infected intermediate or paratenic hosts. However, the ingestion of fresh vegetables contaminated by snail mucus and/or water has also been proposed as a source of the infection for some zoonotic metastrongyloids (e.g., Angiostrongylus cantonensis). In the meantime, the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior are increasingly spreading among cat populations, along with their gastropod intermediate hosts. The aim of this study was to assess the potential of alternative transmission pathways for A. abstrusus and T. brevior L3 via the mucus of infected Helix aspersa snails and the water where gastropods died. In addition, the histological examination of snail specimens provided information on the larval localization and inflammatory reactions in the intermediate host. Methodology/Principal Findings Twenty-four specimens of H. aspersa received ~500 L1 of A. abstrusus and T. brevior, and were assigned to six study groups. Snails were subjected to different mechanical and chemical stimuli throughout 20 days in order to elicit the production of mucus. At the end of the study, gastropods were submerged in tap water and the sediment was observed for lungworm larvae for three consecutive days. Finally, snails were artificially digested and recovered larvae were counted and morphologically and molecularly identified. The anatomical localization of A. abstrusus and T. brevior larvae within snail tissues was investigated by histology. L3 were detected in the snail mucus (i.e., 37 A. abstrusus and 19 T. brevior) and in the sediment of submerged specimens (172 A. abstrusus and 39 T. brevior). Following the artificial digestion of H. aspersa snails, a mean number of 127.8 A. abstrusus and 60.3 T. brevior larvae were recovered. The number of snail sections positive for A. abstrusus was higher than those for T. brevior. Conclusions Results of this study indicate that A. abstrusus and T. brevior infective L3 are shed in the mucus of H. aspersa or in water where infected gastropods had died submerged. Both elimination pathways may represent alternative route(s) of environmental contamination and source of the infection for these nematodes under field conditions and may significantly affect the epidemiology of feline lungworms. Considering that snails may act as intermediate hosts for other metastrongyloid species, the environmental contamination by mucus-released larvae is discussed in a broader context.

Transstadial transmission of Hepatozoon canis from larvae to nymphs of Rhipicephalus sanguineus

Hepatozoon canis is an apicomplexan parasite of dogs, which is known to become infected by ingesting Rhipicephalus sanguineus adult ticks. To investigate the possibility of H. canis transovarial and transstadial transmission from larvae to nymphs, engorged adult female ticks were collected from a private animal shelter in southern Italy, where H. canis infection is highly prevalent. Female ticks (n=35) and egg batches were tested by PCR for H. canis. All eggs examined were PCR-negative whereas 88.6% of females from the environment tested positive. Additionally, fed larvae (n=120) from a dog naturally infected by H. canis were dissected at different time points post collection (i.e. 0, 10, 20 and 30 days). Molted nymphs dissected at 20 days post collection revealed immature oocysts displaying an amorphous central structure in 50% of the specimens, and oocysts containing sporocysts with sporozoites were found in 53.3% of the nymphs dissected at 30 days post collection. This study demonstrates that H. canis is not transmitted transovarially, but it is transmitted transstadially from larvae to nymphs of R. sanguineus and develops sporozoites in oocysts that may infect dogs.

Experimental evidence against transmission of Hepatozoon canis by Ixodes ricinus

Hepatozoon canis is among the most widespread tick-borne protozoa infecting domestic and wild carnivores. Its distribution is related to the occurrence of its major vector, the brown dog tick Rhipicephalus sanguineus. However, the role of Ixodes ricinus as a vector of H. canis has been hypothesized. In the present study, the development of H. canis was investigated in I. ricinus and R. sanguineus nymphs collected from a naturally infested dog. All I. ricinus ticks examined (n=133) were negative by cytological examination at days 20, 30, and 90 post collection, although H. canis DNA was detected in one nymph at day 20 and in 2 nymphs at day 30 post collection. On the other hand, H. canis sporogony was documented by cytology, and H. canis DNA was detected by PCR in R. sanguineus at day 30 post collection. These results indicate that H. canis sporogony does not occur in I. ricinus, but in R. sanguineus, suggesting that I. ricinus does not act as a vector of H. canis.

Detection of Anaplasma platys in dogs and Rhipicephalus sanguineus group ticks by a quantitative real-time PCR

Anaplasma platys is an obligate intracellular, tick-borne pathogen of dogs, which causes canine infectious cyclic thrombocytopenia (CICT). The vector role of Rhipicephalus sanguineus group ticks has been only suggested, but definitive evidence is lacking. This study aimed to detect and quantify A. platys DNA in infected dogs and in their respective ticks through a quantitative real-time PCR assay. From March to May 2009, blood and tick samples from dogs residing in a CICT-endemic area were collected and molecularly analysed. Differences (p<0.05) were detected in the bacterial load between tick nymphs (3.5 × 10(-2)± 2.5 × 10(-2)) and adults (female: 1.2 × 10(-1) ± 1.1 × 10(-1); male: 9.3 × 10(-2)± 1.2 × 10(-2)) and between unengorged (1.1 × 10(-1) ± 2.8 × 10(-2)) and partially or fully engorged ticks (1.9 × 10(-1) ± 2.7 × 10(-2)). No difference was found between ticks collected from positive (1.1 × 10(-1)± 0.6 × 10(-1)) and negative (1.2 × 10(-1) ± 0.3 × 10(-1)) dogs (p>0.05). The mean bacterial load detected in positive dogs was lower than that in their respective ticks (p>0.05). This study provides circumstantial evidence of the putative role of Rhipicephalus sp. I as a vector of this pathogen.

Feline lungworms unlock a novel mode of parasite transmission

Snail-borne lungworms exert an enormous toll on the health and welfare of animals and humans. Of these parasites, Aelurostrongylus abstrusus and Troglostrongylus brevior affect the respiratory tract of felids. These lungworms share both the ecological niche and the species of snail (Helix aspersa) acting as intermediate host. Recently, the ability of H. aspersa to shed infective third-stage larvae (L3s) of A. abstrusus and T. brevior in the environment has been demonstrated, matching previous knowledge of mode of transmission of zoonotic lungworms. Here, we evaluated, for the first time, the ability of A. abstrusus and T. brevior L3s to infect new, susceptible snail hosts following their release from experimentally infected molluscs, and refer to this novel route of parasite transmission as intermediesis. The implications of snail-to-snail transmission in the epidemiology of snail-borne diseases are also discussed.

Cutaneous distribution and localization of Cercopithifilaria sp. microfilariae in dogs.

The aim of this study was to investigate the anatomical distribution of microfilariae of a recently described Cercopithifilaria sp. and the relationship with the preferred attachment sites of its vector, the brown dog tick Rhipicephalus sanguineus. Skin samples from 20 dogs were collected from eight anatomical sites and soaked twice in saline solution at 37°C. All samples were also molecularly processed for the specific amplification of partial cytochrome c oxidase 1 gene. Microfilariae were unevenly distributed on the body, with higher frequencies on interscapular region (n=13; 68.4%) and on the head (n=9; 47.4%). Larval abundance in dogs was positively correlated with the number of positive anatomical sites with a higher percentage of skin samples scoring positive at the microscopic examination of the first sediment (n=47; 30.9%) than of the second (n=8; 5.3%). Histological examination revealed that microfilariae were scattered in the dermis, in association with inflammatory cells. Molecular analysis of skin samples showed a lower frequency of positive sites (n=37; 24.3%) when compared to the microscopic examination (n=53; 34.9%) with five haplotypes sequenced. The results here presented suggested a close co-evolution of this filarioid with its vector.

Pathological and histological findings associated with the feline lungworm Troglostrongylus brevior

Troglostrongylus brevior is a neglected feline lungworm species, which has been increasingly reported in the Mediterranean area, although scant data are available on the respiratory alterations it causes in cats. Therefore, we describe the gross and histological lesions of a 20-week old kitten that succumbed due to the onset of a fulminant respiratory failure. At necropsy, a catarrhal exudate was observed in the airways, along with nematodes in the trachea and bronchi. The lungs were processed for histological examination and serial pulmonary sections were performed. A total of 14 nematodes were collected, being all morphologically and molecularly identified as T. brevior. Lungworms were histologically localized within the bronchial lumen, surrounded by an eosinophilic infiltrate. The presence of T. brevior in the airways has been histologically documented for the first time and its life-threatening potential is discussed.

Apparent tick paralysis by Rhipicephalus sanguineus (Acari: Ixodidae) in dogs

Certain tick species including Ixodes holocyclus can inoculate neurotoxins that induce a rapid, ascending flaccid paralysis in animals. Rhipicephalus sanguineus, the most widespread tick of dogs, is recognized as a vector of several pathogens causing diseases in dogs and humans. A single report suggests its role as cause of paralysis in dogs. This study presents the clinical history of 14 young dogs heavily infested by R. sanguineus (intensity of infestation, 63-328) in an endemic area of southern Italy. During May to June of 2011, dogs were presented at the clinical examination with neurological signs of different degrees (e.g., hind limb ataxia, generalized lethargy, and difficulty in movements). All animals were treated with acaricides and by manual tick removal but ten of them died within a day, displaying neurological signs. The other 4 dogs recovered within 3 days with acaricidal and supportive treatment. Twelve dogs were positive by blood smear examination for Hepatozoon canis with a high parasitemia, two also for Babesia vogeli and two were negative for hemoparasites. Low-grade thrombocytopenia, hypoalbuminemia, and pancytopenia were the haematological alterations most frequently recorded. Other causes of neurological disease in dogs were excluded and the diagnosis of tick paralysis by R. sanguineus was confirmed (ex juvantibus) by early and complete recovery of 4 dogs following acaricidal treatment and tick removal.