Genetic analysis of Cryptozona siamensis (Stylommatophora, Ariophantidae) populations in Thailand using the mitochondrial 16S rRNA and COI sequences.

Cryptozona siamensis, one of the most widespread land snails, is native to Thailand, and plays a key role as an agricultural pest and intermediate host for Angiostrongylus spp. However, its genetic diversity and population structure has not yet been investigated, and are poorly understood. Therefore, a genetic analysis of the C. siamensis population in Thailand was conducted, based mitochondrial 16S rRNA (402 bp) and COI (602 bp) gene fragment sequences. Cryptozona siamensis randomly collected from 17 locations in four populations across Thailand, between May 2017 and July 2018. Fifty-eight snails were used to examine the phylogeny, genetic diversity, and genetic structure. The maximum likelihood tree based on the 16S rRNA and COI fragment sequences revealed two main clades. A total of 14 haplotypes with 44 nucleotide variable sites were found in the 16S rRNA sequences, while 14 haplotypes with 57 nucleotide variable sites were found in the COI sequences. The genetic diversity of C. siamensis in term of the number of haplotypes and haplotype diversity, was found to be high but the nucleotide diversity showed low levels of genetic differentiation for the COI sequence as also noted with the 16S rRNA sequence. The population genetic structure of C. siamensis revealed genetic difference in most populations in Thailand. However, low genetic difference in some populations may be due to high gene flow. This study provides novel insights into the basic molecular genetics of C. siamensis.

Abdominal angiostrongyliasis: pathologic findings in Swiss mice infected with different doses of Angiostrongylus costaricensis.

Abdominal angiostrongyliasis is caused by Angiostrongylus costaricensis, the definitive and intermediate hosts of which are wild rodents and terrestrial molluscs, respectively. Humans are accidental hosts and can be infected by ingesting the third-stage (infective) larvae (L3). It remains unclear whether the number of L3 inoculated is related to lesion severity. Our aim was to analyse histopathological alterations in Swiss mice infected with different doses of A. costaricensis. Thirty-two mice were randomly divided into four groups (n = 8/group): uninfected, control mice; mice infected with a low dose (five L3); mice infected with an intermediate dose (15 L3); and mice infected with a high dose (30 L3). The frequency of intestinal thrombi, splenitis, eggs/larvae, hepatic infarction and acute pancreatitis differed among the groups, the last being considered a significant finding. We conclude that different infective doses alter the histopathological aspects of the infection in Swiss mice, those aspects being more pronounced at medium and high doses, with no effect on the development of the disease. This experimental model shows that the parasite life cycle can be maintained in Swiss mice through the inoculation of a low dose (five L3).

Susceptibility of Biomphalaria glabrata submitted to concomitant infection with Angiostrongylus costaricensis and Schistosoma mansoni / Suscetibilidade de Biomphalaria glabrata submetida à infecção concomitante com Angiostrongylus costaricensis e Schistosoma mansoni

Abstract The easy adaptation of Angiostrongylus costaricensis, nematode responsible for abdominal angiostrongyliasis to several species of terrestrial and freshwater molluscs and the differences observed in the interactions of trematodes with their intermediate hosts have induced us to study the concomitant infection of Biomphalaria glabrata with Schistosoma mansoni and A. costaricensis. Prior exposure of B. glabrata to A. costaricensis (with an interval of 48 hours), favored the development of S. mansoni, observing higher infection rate, increased release of cercariae and increased survival of molluscs, when compared to molluscs exposed only to S. mansoni. Prior exposure of B. glabrata to A. costaricensis and then to S. mansoni also enabled the development of A. costaricensis since in the ninth week of infection, higher amount of A. costaricensis L3 larvae was recovered (12 larvae / mollusc) while for molluscs exposed only to A. costaricensis, the number of larvae recovered was lower (8 larvae / mollusc). However, pre-exposure of B. glabrata to S. mansoni (with an interval of 24 hours), and subsequently exposure to A. costaricensis proved to be very harmful to B. glabrata, causing extensive mortality of molluscs, reduced pre-patent period to release cercariae and greater recovery of L3 A. costaricensis larvae.

Resumo A facilidade de adaptação do Angiostrongylus costaricensis, nematódeo responsável pela angiostrongiliase abdominal, a diversas espécies de moluscos terrestres e dulciaquícolas e as divergências observadas nas interações dos trematódeos com seus hospedeiros intermediários nos induziu a estudar a infecção concomitante de Biomphalaria glabrata com Schistosoma mansoni e A. costaricensis. A exposição prévia de B. glabrata ao A. costaricensis (com intervalo de 48 horas), favoreceu o desenvolvimento do S. mansoni observando-se elevação da taxa de infecção, maior liberação de cercárias e maior sobrevivência dos moluscos, quando comparado com os moluscos expostos somente ao S. mansoni. A exposição de B. glabrata previamente ao A. costaricensis e posteriormente ao S. mansoni também facilitou o desenvolvimento do A. costaricensis uma vez que na nona semana de infecção foi recuperada maior quantidade de larvas L3 de A. costaricensis, enquanto nos moluscos expostos somente ao A. costaricensis, o número de larvas recuperadas foi menor . Entretanto a pré-exposição de B. glabrata ao S. mansoni (com intervalo de 24 horas), e posteriormente a exposição ao A. costaricensis mostrou-se muito prejudicial à B. glabrata provocando grande mortalidade dos moluscos, redução do período pré-patente para liberação de cercárias e maior recuperação de larvas L3 de A. costaricensis.

Susceptibility of Biomphalaria glabrata submitted to concomitant infection with Angiostrongylus costaricensis and Schistosoma mansoni.

The easy adaptation of Angiostrongylus costaricensis, nematode responsible for abdominal angiostrongyliasis to several species of terrestrial and freshwater molluscs and the differences observed in the interactions of trematodes with their intermediate hosts have induced us to study the concomitant infection of Biomphalaria glabrata with Schistosoma mansoni and A. costaricensis. Prior exposure of B. glabrata to A. costaricensis (with an interval of 48 hours), favored the development of S. mansoni, observing higher infection rate, increased release of cercariae and increased survival of molluscs, when compared to molluscs exposed only to S. mansoni. Prior exposure of B. glabrata to A. costaricensis and then to S. mansoni also enabled the development of A. costaricensis since in the ninth week of infection, higher amount of A. costaricensis L3 larvae was recovered (12 larvae / mollusc) while for molluscs exposed only to A. costaricensis, the number of larvae recovered was lower (8 larvae / mollusc). However, pre-exposure of B. glabrata to S. mansoni (with an interval of 24 hours), and subsequently exposure to A. costaricensis proved to be very harmful to B. glabrata, causing extensive mortality of molluscs, reduced pre-patent period to release cercariae and greater recovery of L3 A. costaricensis larvae.

Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.

This study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.

Eosinophilic meningitis.

Eosinophilic meningitis is defined by the presence of at least 10% eosinophils in the total cerebrospinal fluid (CSF) leukocyte count. Although there are several possible causes of eosinophils in the CSF, parasitic infection is the main cause. The three common parasites causing eosinophilic meningitis include Angiostrongylus cantonensis, Gnathostoma spinigerum, and Taenia solium. Even though these parasites are endemic in tropical countries, they are now spreading globally due to extensive traveling, and physicians worldwide should pay more attention to this condition. This chapter will review risk factors, clinical manifestations, and treatment of these three parasites.

Effect of temperature on activity of third-stage larvae of Angiostrongylus vasorum.

To evaluate the effect of temperature on the activity and mortality of the L3 of Angiostrongylus vasorum, 1,500 L3 were isolated from experimentally infected snails and distributed into five equal groups. Three groups were incubated at 37°C, 27°C, and 5°C. The remaining two groups were incubated at 27°C and 5°C for 10 days, at which time the temperature for the 27°C group was reduced to 5°C and the 5°C group increased to 27°C. Larva activity was observed daily and inactive larvae were removed. At 37°C, larvae survived up to 8 days. At 27°C, larvae were active until day 6. When subjected to a reduction in temperature from 27°C to 5°C beginning on day 10, the number of active larvae increased until day 13. Only on day 17 did the number of active larvae decline to zero. At 5°C, larvae remained active until day 15, surviving to 24 days. When temperature was increased from 5°C to 27°C beginning on day 10, larvae were found active until day 12 and maintained an intermediate level of activity to day 21. Survival of larvae was greater at lower temperatures, while high temperatures were associated with higher mortality.

Canine and feline infections by cardiopulmonary nematodes in central and southern Italy.

Capillaria aerophila, Aelurostrongylus abstrusus, Angiostrongylus vasorum and Dirofilaria immitis are cardiopulmonary nematodes affecting dogs and cats and presently emerging in several countries. The results obtained in 2009 - 2010 during a study aiming to investigate the occurrence of these nematodes in regions from Central (Marche and Abruzzo regions--Sites A and B, respectively) and southern (Apulia--Site C) Italy are here reported. A total of 534 and 436 individual faecal samples collected from dogs and cats were examined, together with 471 and 34 faecal environmental samples taken from dog shelters and catteries. One hundred and ninety-two individual blood samples were also collected from dogs. Faeces were examined using copromicroscopical flotations and Baermann technique, whereas blood samples were tested by Knott's method. Eggs of C. aerophila were detected in 1.48 % and 20 % (Site A), 8.67 % and 2.71 % (Site B), and 16.67 % and 0 % (Site C) of individual and environmental canine samples. C. aerophila was found in 2.90 % (Site A), 3.03 % (Site B) and 14.29 % (Site C) of individual cats. Larvae of A. vasorum were found in 0.96 % and 2.48 % of individual and environmental samples from Site B, respectively, while those of A. abstrusus in 1.82 % (Site A) and 9.96 % (Site B) of individual faeces. Microfilariae of Dirofilaria immitis, identified on the basis of key morphological and morphometric features, were detected in 2.56 % of samples collected from Site B. Despite the small sample size, these results indicate that cardiopulmonary nematodes occur in Central and southern Italy, thus they should be included in the differential diagnosis of pet cardiorespiratory diseases. Larger surveys are necessary to gain more information on the diffusion of these parasites, especially for C. aerophila and A. vasorum, for which the actual distribution is poorly known.

Canine angiostrongylosis: the French heartworm: an emerging threat in North America.

Angiostrongylus vasorum, French heartworm, is a metastrongloid parasite found in the pulmonary arteries and right ventricle of wild and domestic canids and various other animals. The natural definitive hosts are species of foxes. The geographic distribution of the parasite includes various countries of Europe, Africa, South America, and North America. Angiostrongylosis is considered an emerging disease in dogs in Europe. In North America, autochthonous A. vasorum infection occurs only in the Canadian province of Newfoundland-Labrador. Computer modeling suggests there is a high probability that A. vasorum will spread to other parts of North America and will likely become endemic in the eastern half of the continent and in the states and provinces along the western coast. Animals acquire infection by the ingestion of gastropod or frog intermediate hosts that carry the infective 3rd-stage larvae. Frogs can also serve as paratenic hosts. Definitive antemortem diagnosis is by detection of L(1) in feces, sputum, or bronchoalveolar lavage samples. Baermann fecal examination is the most reliable method for fecal detection. However, false negative results can occur due to the typical erratic/sporadic fecal larval shedding pattern of A. vasorum. Recently, promising new methods for A. vasorum infection diagnosis have been reported involving polymerase chain reaction of blood and fecal samples and a sandwich ELISA for detection of circulating worm excretory/secretory antigen. Current treatment options include moxidectin, milbemycin oxime, and fenbendazole.

Susceptibility and morbidity between male and female Swiss mice infected with Angiostrongylus costaricensis.

The gender of vertebrate hosts may affect the outcome of parasitic infections. An experimental murine infection with Angiostrongylus costaricensis was followed with determinations of body weight, fecal larval elimination, number and length of adult worms, number of macroscopic intestinal lesions, and mortality. Groups of male and female Swiss mice were infected with 10 3(rd)-stage A. costaricensis larvae per animal. The results indicate there are no significant differences related to gender of the host, except for higher length of worms developed in male mice.

Angiostrongylus vasorum infection in dogs: continuing spread and developments in diagnosis and treatment.

Disease caused by Angiostrongylus vasorum is increasingly diagnosed in dogs, as the geographic range of the parasite increases along with awareness among clinicians. Diagnosis, treatment and prevention are not always straightforward, although recent developments offer hope for improved options in future. Understanding of the epidemiology and pathogenesis remains poor. This paper provides an overview of the current state of knowledge of this parasitic disease, focussing on the most recent developments and advances.

Susceptibility and morbidity between male and female Swiss mice infected with Angiostrongylus costaricensis: Susceptibilidade e morbidade entre camundongos Swiss machos e fêmeas infectados com Angiostrongylus costaricensis

The gender of vertebrate hosts may affect the outcome of parasitic infections. An experimental murine infection with Angiostrongylus costaricensis was followed with determinations of body weight, fecal larval elimination, number and length of adult worms, number of macroscopic intestinal lesions, and mortality. Groups of male and female Swiss mice were infected with 10 3rd-stage A. costaricensis larvae per animal. The results indicate there are no significant differences related to gender of the host, except for higher length of worms developed in male mice.

O sexo dos hospedeiros vertebrados pode influenciar no resultado de infecções parasitárias. A infecção experimental de camundongos com Angiostrongylus costaricensis foi acompanhada com observação do peso corporal, eliminação de larvas nas fezes, número e comprimento dos vermes adultos, número de lesões macroscópicas nos intestinos e mortalidade. Grupos de camundongos Swiss machos e fêmeas foram infectados cada um com 10 larvas de terceiro estágio de A. costaricensis. Os resultados indicam que não há diferenças significativas relacionados ao sexo dos hospedeiros, exceto pelo maior comprimento dos vermes nos hospedeiros machos.

Clinical, laboratory and pathological findings in dogs experimentally infected with Angiostrongylus vasorum.

The aim of this comparative study was to investigate the development of clinical signs and accompanying haematological, coproscopic and pathological findings as a basis for the monitoring of health condition of Angiostrongylus vasorum infected dogs. Six beagles were orally inoculated with 50 (n=3) or 500 (n=3) A. vasorum third stage larvae (L3) obtained from experimentally infected Biomphalaria glabrata snails. Two dogs were treated with moxidectin/imidacloprid spot-on solution and two further dogs with an oral experimental compound 92 days post infection (dpi), and were necropsied 166 dpi. Two untreated control dogs were necropsied 97 dpi. Prepatency was 47-49 days. Dogs inoculated with 500 L3 exhibited earlier (from 42 dpi) and more severe respiratory signs. Clinical signs resolved 12 days after treatment and larval excretion stopped within 20 days in all four treated dogs. Upon necropsy, 10 and 170 adult worms were recovered from the untreated dogs inoculated with 50 and 500 L3, respectively. Adult worms were also found in two treated dogs, in the absence of L1 or eggs. Despite heavy A. vasorum infection load and severe pulmonary changes including vascular thrombosis, only mild haematological changes were observed. Eosinophilia was absent but the presence of plasma cells was observed. Neutrophilic leucocytes showed a transient increase but only after treatment. Signs for coagulopathies were slight; nevertheless coagulation parameters were inoculation dose dependent. Ten weeks after treatment pulmonary fibrosis was still present. Infections starting from 50 L3 of A. vasorum had a massive impact on lung tissues and therefore on the health of affected dogs, particularly after prepatency, although only mild haematological abnormalities were evident.

Angiostrongylus vasorum: the 'French Heartworm'.

Angiostrongylus vasorum which is commonly known as 'French heartworm' is a snail-born parasitic disease affecting the members of the Canidae family. This parasite has a cosmopolitan distribution covering tropical, subtropical and temperate regions. However, its distribution is characterised by isolated endemic foci, with only sporadic occurrences outside this areas. During the last two decades, several sporadic occurrences in old and new endemic areas have been documented by the researchers. However, the spread of infection and dynamic consequences of this parasite in final host has not been clarified yet. Therefore, this review will focus on the morphology, biology, clinical significant as well as management of this parasitic disease.

[Angiostrongylosis or eosinophilic meningitis]. / Angiostrongyloïdose ou méningite à éosinophiles.

Eosinophilic meningitis, or angiostrongyliasis, is a common disease in Asia, in the Caribbean and in the Pacific islands. It is caused by a rat lungworm Angiostrongylus cantonensis. Infection occurs by consumption of raw or undercooked snails. Diagnosis is based on epidemiological criteria, clinical manifestations, elevated count of eosinophils in the cerebrospinal fluid and serological tests. Treatment is symptomatic and supportive.

The effect of temperature and host age on the infectivity and development of Angiostrongylus vasorum in the slug Arion lusitanicus.

Experimental infection with Angiostrongylus vasorum was conducted in Iberian slugs Arion lusitanicus. Initially, different size/age groups of juvenile slugs (small, <0.5 g and medium, 0.5-1 g) were exposed to freshly isolated first-stage parasitic larvae (L1) of A. vasorum. The slugs were subsequently incubated at 5, 10 and 15 degrees C for 6 weeks. Larval development within the slugs differed significantly with temperature. At 15 degrees C, all larvae developed into the third larval stage (L3), at 10 degrees C into the second stage (L2), whereas no development was observed at 5 degrees C. The mean larval burdens were highest in the largest group of slugs and tended to increase with higher temperature. In a second experiment isolated L1 were incubated at 5, 10 and 15 degrees C for 3 and 7 days prior to infection of slugs, which then were kept for 6 weeks at 15 degrees C. The infectivity decreased significantly with the larval storage time and the mean larval burden per slug was lower at higher incubating temperature. However, all established larvae developed into infective L3. Temperature had an effect on the development of the larvae and thus an impact on transmission of the parasite as only L3 are infective to the definitive canid hosts.

Canine angiostrongylosis: an emerging disease in Europe.

OBJECTIVE: The aim of this article is to review Angiostrongylus vasorum infection in dogs, including the life cycle, signalment, clinical signs, diagnosis, and treatment. Apparent changes in the epidemiology of this unique parasite are considered, alongside information available regarding its recent geographic spread. ETIOLOGY: A. vasorum is a metastrongyloid parasite capable of causing an array of clinical problems in dogs, including cardiorespiratory, coagulopathic, and neurologic signs. Currently, the parasite has a worldwide distribution; however, it usually arises in small pockets of enzootic foci. Recent reports suggest a changing distribution of this parasite, which has renewed interest in its epidemiology and in the risk of expansion to new areas including mainland North America. DIAGNOSIS: A definitive diagnosis of angiostrongylosis is usually made using the modified Baermann technique either using feces or tracheobronchial secretions; however, this review also discusses novel methods such as serologic and molecular techniques. THERAPY: Once a diagnosis of angiostrongylosis is made, prompt treatment should follow with anthelmintic drugs (such as moxidectin/imidacloprid, milbemycin oxime, or fenbendazole) and supportive care dependent upon the patient's clinical signs. Currently, there is no proven prophylactic regime. PROGNOSIS: The prognosis appears to be very dependent upon the severity of clinical signs at presentation. A. vasorum can be fatal and death may be sudden. However, if a prompt diagnosis is made and appropriate treatment is administered complete clinical resolution is possible.

Quantitative computed tomography evaluation of pulmonary disease.

Objective assessment of pulmonary disease from computed tomography (CT) examinations is desirable but difficult. When such assessments can be made, it is important that they are related to some part of the pathophysiologic process present. Herein we propose that automated volume histogram analysis can yield data that allow differentiation of normal from abnormal lung, and that the magnitude of disease will have an association with objective CT indices. Data from pulmonary CT images from 34 foxes (six uninfected controls and 28 infected with Angiostrongylus vasorum, subdivided by age and infective dose) were available. Lung tissue was segmented from surrounding tissue using an automated segmentation method. A volume histogram showing voxel frequency for each CT number in the range -1024 to -250 HU was created from the entire image stack from each fox. Using these data, the inter-quartile range and the CT number at the 95th percentile were determined. The results showed that segmentation could be readily achieved but that areas of severely diseased lung were excluded. Based on two-way analysis of variance for both the inter-quartile range and the CT number at the 95th percentile, both quantities were significantly affected by the infection status of the animal and were related to worm burden (P < 0.001). The study shows that this form of analysis is readily achieved and provides quantitative data that can be used to assess disease severity, progression, and response to treatment.

Angiostrongylus costaricensis (Nematoda: Protostrongylidae): migration route in experimental infection of Omalonyx sp. (Gastropoda: Succineidae).

Angiostrongylus costaricensis can infect several mollusks, and its migration route in intermediate hosts has been studied only in Sarasinula marginata. To verify the susceptibility of Omalonyx sp. as an intermediate host of A. costaricensis and to analyze the nematode migration route, individuals were infected with stage 1 larvae. Obtained stage 3 larvae were orally inoculated in mice, and after 30 days, adult worms and stage 1 larvae were recovered, demonstrating Omalonyx susceptibility and suitability to infection. To define the parasite migration routes, specimens of Omalonyx with 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 2 days, 5 days, 10 days, 12 days, 15 days, 20 days, 21 days, 25 days, 28 days, and 30 days of infection were fixed and serially sectioned. Histological sections were stained with hematoxylin-eosin. The results were compared to those described in S. marginata. Oral and cutaneous infections were noted. After the penetration, larvae were retained, mainly in the fibromuscular tissue, by hemocytes, or they spread to the whole organism through the circulation, following the anatomical structure of the vasculature. The perilarval hemocyte reaction in Omalonyx was more intense until stage 2 larva instar, decreasing in the presence of stage 3 larvae. Differences in some aspects of hemocyte reaction between S. marginata and Omalonyx exemplify interspecific peculiarities in snail response to the same parasite.