New host, geographic records, and histopathologic studies of Angiostrongylus spp (Nematoda: Angiostrongylidae) in rodents from Argentina with updated summary of records from rodent hosts and host specificity assessment

To date, 21 species of the genus Angiostrongylus (Nematoda: Angiostrongylidae) have been reported around the world, 15 of which are parasites of rodents. In this study, new host, geographic records, and histopathologic studies of Angiostrongylus spp in sigmodontine rodents from Argentina, with an updated summary of records from rodent hosts and host specificity assessment, are provided. Records of Angiostrongylus costaricensis from Akodon montensis andAngiostrongylus morerai from six new hosts and geographical localities in Argentina are reported. The gross and histopathologic changes in the lungs of the host species due to angiostrongylosis are described. Published records of the genus Angiostrongylus from rodents and patterns of host specificity are presented. Individual Angiostrongylusspecies parasitise between one-19 different host species. The most frequent values of the specificity index (STD) were between 1-5.97. The elevated number of host species (n = 7) of A. morerai with a STD = 1.86 is a reflection of multiple systematic studies of parasites from sigmodontine rodents in the area of Cuenca del Plata, Argentina, showing that an increase in sampling effort can result in new findings. The combination of low host specificity and a wide geographic distribution of Angiostrongylus spp indicates a troubling epidemiological scenario although, as yet, no human cases have been reported.

siensis, A. siamensis, and A. schmidti. In those species inhabiting the pulmonary arteries, eggs deposited by adults develop to first stage larvae in the lungs which move up the airways, are swallowed and pass in the faeces. This developmental pathway is exemplified by A. andersoni, A. dujardini, and A. schmidti (Kinsella 1971, Bhaibulaya 1975, Mota & Lenzi 2005, Spratt 2015. The resultant pathology has been described in species such as A. cantonensis, A. costaricensis, A. mackerrasae, A. morerai, A. sandarsae, A. schmidti, and A. siamensis (Mackerras & Sandars 1955, Alicata 1968, Kinsella 1971, Tesh et al. 1973, Ohbayashi et al. 1979, Mota & Lenzi 2005, Robles et al. 2012. One of the most important properties characterising a parasite taxon is its host specificity. It is indicative of intrinsic biological characteristics of both host and parasite and an emergent property of their ecological and evolutionary relationship (Dick & Patterson 2007). Host specificity can be defined as the extent to which a parasite taxon is restricted in the number of host species used at a given stage in the life cycle (Poulin 2007).
In this paper, we provide new host and geographical records for two species of Angiostrongylus from sigmodontine rodents in Argentina and describe the gross and histopathologic changes in the lungs of the host species due to angiostrongylosis. Moreover, we present comprehensive data on all the records of the genus Angiostrongylus from rodents and evaluate patterns of host specificity.

MATERIALS AND METHODS
Cricetid rodents were trapped during different field studies between 2007-2012 (see acknowledgements and financial support) and the following species were examined for angiostrongylid nematodes: eight specimens of Deltamys kempi Thomas 1917 from Reserva Natural de la Costanera Sur (34º36'S 58º27'W), Ciudad Autónoma de Buenos Aires and La Balandra (34º56'S 57º42'W), Partido de Berisso, province of Buenos Aires, 11 specimens of Akodon montensis Thomas 1913 from RP2, 6 km NE, Arroyo Paraíso, (27º12'47.7"S 54º01'59.9"W)  The viscera (included lungs) were fixed whole in 10% buffered formalin and examined. Pulmonary arteries and veins were opened and observed for adult worms using a stereoscopic microscope. Adult nematodes were collected, preserved in 70% ethanol, cleared in lactophenol, and studied under a light microscope. Drawings were made with the aid of a drawing tube. Each of the five lobes of the lungs was trimmed in the subterminal transversal part, processed, sectioned at 5 µm (± 25 sections per slide), stained with haematoxylin and eosin (H&E), and examined microscopically.
Quantitative parameters of prevalence (P = specimens parasitised/specimens examined x 100) was calculated according to Bush et al. (1997) for each host species and locality.
Records of species of Angiostrongylus from rodents were compiled from the literature (scientific papers and book sections). When necessary, scientific names of mammal hosts have been updated following Edwards et al. (1993), Wilson and Reeder (2005), Weksler et al. (2006), and Srinivasulu and Srinivasulu (2011). In order to evaluate host specificity, the specificity index (S TD ) by Poulin and Mouillot (2003) was calculated. This index measures the average taxonomic distinctness of all host species used by a parasite species. All mammal species included were fitted into a taxonomic structure with six hierarchical levels above species, i.e., genus, subfamily, family, superfamily, order, and class (Mammalia). The range of index can vary between 1-6, and since the index cannot be computed for parasites exploiting a single host species, the value of zero is assigned to reflect strict host specificity. The value of this index is inversely proportional to host specificity. The asymmetries in the taxonomic distribution of host species were calculated through variance in taxonomic distinctness (VarS TD ) (Poulin & Mouillot 2003). A record was defined as the finding of a parasite species on a definitive host and, at a given locality, regardless of the number of host sampled and of nematodes collected on a particular host. The aberrant host species reported (Maldonado et al. 2012, Spratt 2015 which showed signs of disease were included in the calculation of host specificity, but not the experimentally infected or accidental host species.
Adult specimens and H&E stained sections (slides) of lung were deposited in the Helminthological Collection of the Museo de La Plata (CHMLP A. costaricensis 7052 and A. morerai 7053-7059, respectively) and the hosts were deposited in the Mastozoological Collections of the Centro Nacional Patagónico (CNP 1968, 2338, Puerto Madryn, Chubut, Argentina. Ethics -The research has been conducted according to Argentine laws. Sample collection was carried out during fieldwork under official permits granted by Fauna and Flora of the Province of Buenos Aires (expedient 22500-7981/10), Ministry of Industry and Environment of the Province of Formosa (authorisation n/n; transit guide: 004076), Ministry of Ecology, Renewable Natural Resources, and Tourism of Misiones (authorisation 24 and 27, transit guides: 000316 and 000371). This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The specimens obtained with methods for live capture were studied and humanely sacrificed following the procedures and protocols approved by national laws (Animal Protection National law 14.346 and references in the provincial permits) and Ethical Committee for Research on Laboratory Animals, Farm, and Obtained from Nature of National Council of Scientific and Technical Research (resolution 1047, section 2, annex II), and subsequently by National Agency for the Promotion of Science and Technology of Argentina (PICT 2010-0924). No endangered species were involved in this study.

RESULTS
A single male specimen of Angiostrongylus found in the caecal mesenteric arteries of A. montensis from El Soberbio was identified as A. costaricensis. Adult specimens found in the pulmonary arteries and heart of D. kempi from La Balandra and Reserva Natural de la Costanera Sur, A. montensis and S. angouya from Refugio Mocona, and A. azarae, C. callosus, and N. lasiurus from Reserva El Bagual were identified on the basis of the morphology of the bursa, spicules, and diagnostic measurements as A. morerai (Fig. 1). Table I lists prevalence of infections for all hosts examined. The prevalence of A. costaricensis was very low (9%). The highest prevalence of A. morerai was recorded in D. kempi. The region with the most records of this nematode was La Pampa ecoregion (P = 62.5%) in the province of Buenos Aires. The Selva Paranaense (province of Misiones) and Chaco Húmedo (province of Formosa) ecoregions showed similar values (P = 20% and 17.4%, respectively) ( Table I).  Specimens of A. morerai were present in heart chambers ( Fig. 2A) and in pulmonary arteries sometimes showing the complete obliteration of the lumen (Fig. 2B). The infected rodents showed macroscopic lesions (firm nodules) of verminous pneumonia in three, four, or five lobes. Each lung lobe contained multiple small yellowish nodules scattered throughout the parenchyma (Fig. 2C).
Additionally, histopathology examination of tissue fragments showed multiple nodules in the vessels, interstitium, and alveoli. Nodules were formed by larvae surrounded by an elevated number of granulocyte and mononuclear cells (Fig. 2M). The vessels, interstitium, and alveoli contained nematode larvae with mild to moderate interstitial fibrosis (Fig. 2F-M). Worms were approximately 80-200 µm long and contained numerous discrete basophilic and eosinophilic granules (Fig. M). Numerous nodules (set of eggs and larvae) surround by granulomatous reactions were situated under the pleural surface (Fig. 2F, J). Several damaged capillaries and small arterioles were observed (Fig. 2D-H).
The lobe with the greatest intensity of larvae proportionally was the left upper followed by the right lower and right medium lobes, the right upper and left lower lobes, had similar, but smaller, intensities of infection. As estimation about one-five larvae per 200 µm 2 x 5 µm thickness could be observed in the left upper lobe. In the other lobes, the nodules were more scattered. The host with the most nodules (set of larvae) surrounded by granulomatous reactions was C. callosus (Fig. 2J, K).
Number of host species for all Angiostrongylus species found in rodents and values of S TD and VarS TD for each species are shown in Table II and depicted in Fig. 3. The distribution of number of host species was skewed considering only the natural infection by angiostrongylosis (Fig. 3). The figures clearly show that most Angiostrongylus species parasitise between one-19 different host species: five Angiostrongylus species were associated with a single species, A. andersoni and A. sandarsae were found in two host species, A. dujardini and A. mackerrasae in four host species, A. morerai and A. siamensis in six host species, and the rest in more than 10 host species. The values of S TD were between 1-5.97. The value of zero was assigned for five species to reflect the strict host specificity. A. andersoni, A. sandarsae, A. mackerrasae, A. morerai, and A. siamensis parasitise species hosts that belong to different subfamilies (S TD = 1-2), A. dujardini to different families (S TD = 2-3), A. malaysiensis to different superfamilies (S TD = 3-4), and A. cantonensis and A. costaricensis to different orders (S TD = 5-6). With respect to the gross and histopathologic changes in the lungs of the host species, a different degree of pathogenicity was observed among the hosts, with the highest being in C. callosus (Phyllotini). This is the first record of Angiostrongylus sp. in this tribe of sigmodontine rodents. As demonstrated in Robles et al. (2012), in A. morerai, the resulting immune reaction can cause interstitial fibrosis and the destruction of small capillaries and arterioles. In that study and here, extensive lesions were apparently caused by a single male and female ( Fig.  2A, B). Macroscopic lesions of verminous pneumonia in the lungs were similar to those described for A. mackerrasae by Mackerras and Sandars (1955) and A. sandarsae by Alicata (1968). Histopathological examination revealed nodules formed as a result of larvae being surrounded by granulocytes and mononuclear cells (Fig. 2C-M).

Baylis 1928
Apodemus mystacinus cata 1968, Kinsella 1971, Bhaibulaya 1975, Mota & Lenzi 2005, Robles at al. 2012. However, to know the complete pathogenicity and potential transmission of each parasitic species, studies on intermediate hosts and the reaction of the larvae in the affected organs must be completed. The presence of A. morerai in Argentina in different ecoregions indicates that environmental features may have little influence on geographic distribution, although it is interesting to note that apparently these can influence frequency and abundance. Prevalence in the La Pampa ecoregion was considerably higher than Selva Paranaense (20%) and Chaco Húmedo (17.4%). The question is whether the differences in the frequency and abundance of Angiostrongylus spp may be due to the sampling effort and/or to the distribution of definitive and intermediate hosts and/or to the susceptibility of both. For example, with respect to the latter, Combes (2001) proposed different filters of parasite-host association; encounter filters (biodiversity and behaviour) and compatibility filters (resource and density).
Five species of Angiostrongylus have been reported in North and South American rodents: A. cantonensis, A. costaricensis, A. lenzii, A. morerai, and A. schmidti (Teixeira et al. 1993, Díaz et al. 2013. Accordingly, the low host specificity of these Angiostrongylus spp it is puzzling that there been no cases of eosinophilic meningoencephalitis or abdominal angiostrongylosis in Argentina to date. Spratt (2015) partly answer to a similar situation, since the reports in the literature of many species of Angiostrongylus in rodents reflect lack of opportunity or interest in examining nonurban and nonagricultural hosts (Table II).
The elevated number of host species (n = 7) of A. morerai with a S TD = 1.86 is a reflection of multiple systematic studies of parasites from sigmodontine rodents in the area of Cuenca del Plata Argentina, showing that an increase in sampling effort can result in new findings. Therefore, a low number of host species used by other Angiostrongylus species may be an artifact caused by lack of sampling effort. This is the first attempt to describe general patterns of host specificity of Angiostrongylus from rodents through a quantitative approach. Host specificity values did not include the hosts recorded as part of experimental infections or accidental hosts (Table II) (Kinsella 1971, Petter & Cassone 1975, McKenzie et al. 1978, Higgins et al. 1997, Barrett et al. 2002. However, those studies support the conclusions of this survey, since the addition of hosts from other families and orders would only increase the S TD values. In conclusion, the distribution of Angiostrongylus spp shows no environmental limits, demonstrates low host specificity, and indicates that their host range has probably been underestimated. In addition, there are other host records of some species of Angiostrongylus which need to be confirmed by morphological and molecular analysis (Robles et al. 2012). Moreover, it is necessary to explore the different degrees of pathogenicity in various hosts, mainly in those cases that are phylogenetically more distant (different host family) to analyse which are the filters (meeting, immunological, etc.) that determine host distribution. These results would allow anticipating contingencies and prevention planning for diseases caused by angiostrongylosis.
There is a need to increase awareness in the human population about the risk of contracting angiostrongyliasis and healthcare providers should consider these parasites on the South American continent when making medical diagnoses. Moreover, surveillance and control of intermediate and definitive hosts as well as health education should be done to avoid human infections.