Detection of Neospora caninum DNA by polymerase chain reaction in bats from Southern China

Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Domestic dogs and other canids function as definitive hosts, while other mammals serve as natural intermediate hosts. In the present study, the brain tissues of bats collected in Yunnan Province, Southern China were tested by N. caninum specific-nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA to determine whether bats could be infected with N. caninum. N. caninum DNA was detected in 1.8% (4/227) of bats, i.e., 1.7% (1/60) in Rousettus leschenaultia, 1.7% (1/58) in Hipposideros pomona, 2.9% (2/69) in Rhinolophus pusillus, and none (0/40) in Myotis daubentoniid. The findings of the present study are only the first indication that bats could serve as an intermediate host, and further studies are necessary to confirm whether bats are involved in the transmission of N. caninum infections.     

Molecular detection of coccidian Apicomplexa Parasites isolated from wild crab-eating and pampas foxes through novel TaqMan™ probes: a contribution to their molecular epidemiology

Neospora caninum, Toxoplasma gondii and Hammondia spp. are coccidian parasites similar in morphology. Molecular techniques are necessary to detect parasite DNA isolated from stool samples in wild canids because they were reported as definitive hosts of N. caninum life cycle. The objective of this study was to develop a highly sensitive and accurate molecular method for the identification of coccidian Apicomplexa parasites in crab-eating fox (Cerdocyon thous) and pampas fox (Lycalopex gymnocercus). Tissue samples from road-killed animals (pampas fox?=?46, crab-eating fox?=?55) and feces (pampas fox?=?84, crab-eating fox?=?2) were collected, and species were diagnosed through molecular assay. PCR was used for the amplification of a fragment of the coccidian Apicomplexa nss-rRNA gene. Additionally, we developed a novel real-time PCR TaqMan? probe approach to detect T. gondii- Hammondia spp. and N. caninum. This is the first report of N. caninum DNA in pampas fox feces (n?=?1), thus it was also detected from pampas fox tissues (n?=?1). Meanwhile, T. gondii was found in tissues of pampas (n?=?1) and crab-eating (n?=?1) foxes and H. triffittae in one crab-eating fox tissue. Despite the low percentage (2.5%) of positive samples, the molecular method developed in this study proved to be highly sensitive and accurate allowing to conduct an extensive monitoring analysis for these parasites in wildlife.

     

Neospora caninum: detection in wild rabbits and investigation of co-infection with Toxoplasma gondii by PCR analysis

Neospora caninum is an important pathogen of cattle causing significant economic loss. There is much current interest in wild animal reservoirs for this parasite. The role of the rabbit in this is currently unknown. DNA samples from the brains of wild rabbits (Oryctolagus cuniculus) collected from the Malham area of the Yorkshire dales were investigated by species-specific PCR for the presence of N. caninum and Toxoplasma gondii. We found prevalences of N. caninum of 10.5% (6/57) and T. gondii of 68.4% (39/57) with 8.8% (5/57) co-infected. Strain typing of T. gondii positive rabbits revealed strain types I-III were present in this population. Investigation of tissue distribution determined N. caninum DNA was most often detected in the brain and heart, less often in the tongue and not in the liver. To our knowledge this is the first report of N. caninum detection in naturally infected wild rabbits.     

Natural breeding with bulls experimentally infected with Neospora caninum failed to induce seroconversion in dams

Four bulls and 56 heifers seronegative to Neospora caninum were used to determine the feasibility of venereal transmission in bovine neosporosis under natural conditions. Bulls were experimentally infected with 10⁸ live N. caninum tachyzoites. Two of them with the Nc-1 isolate and the other two with the Nc-Spain-7 isolate. After 13 months of initial infection, each bull was re-infected with the same isolate and dose. The experiments were carried out from March to September during 2006 and 2007 where groups of cyclic heifers were naturally mated by the experimentally infected bulls. In year 2006, two bulls infected with different N. caninum isolate serviced 12 heifers each. In year 2007, the same bulls serviced the same heifers a second time (now primiparous) and six new heifers were also added to each group. In addition, the other two bulls serviced 10 additional heifers each. Experimental animals were monitored for 30 weeks and serum samples were collected weekly and fortnightly in years 2006 and 2007, respectively to evaluate the presence of specific antibodies to N. caninum. Experimentally infected bulls showed a significant increase of specific IgG antibodies from 13 (Nc-SP-7) and 21 (Nc-1) days post-infection. Serum IgG antibody responses of individual animals were similar in kinetics but slightly different in magnitude. Serum samples from heifers were all negative. Pregnant rates were 100% in heifers and 91% in primiparous animals. Calves did not show precolostral specific antibodies to N. caninum. Venereal transmission of bovine neosporosis under natural grazing conditions is unlikely to occur.     

Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.     

Role of dense granule antigen 7 in vertical transmission of Neospora caninum in C57BL/6 mice infected during early pregnancy

Neosporosis is a parasitic disease affecting the health of dogs and cattle worldwide. It is caused by Neospora caninum, an obligate intracellular apicomplexan parasite. Dogs are its definitive host, it mostly infects livestock animals, especially cattle that acts as intermediate host. It is necessary to have well-established models of abortion and vertical transmission in experimental animals, in order to determine basic control measures for the N. caninum infection. We evaluated the role of N. caninum dense granule antigen 7 (NcGRA7) in the vertical transmission of N. caninum using the C57BL/6 pregnant mouse model. We inoculated mice on day 3.5 of pregnancy with parental Nc-1 or NcGRA7-deficient parasites (NcGRA7KO). Post-mortem analyses were performed on day 30 after birth and the surviving pups were kept until day 30 postpartum. The number of parasites in the brain tissues of offspring from NcGRA7KO-infected dams was significantly lower than that of the Nc-1-infected dams under two infection doses (1 × 10⁶ and 1 × 10⁵ tachyzoites/mouse). The vertical transmission rates in the NcGRA7KO-infected group were significantly lower than those of the Nc1-infected group. To understand the mechanism by which the lack of NcGRA7 decreases the vertical transmission, pregnant mice were sacrificed on day 13.5 of pregnancy (10 days after infection), although parasite DNA was detected in the placentas, no significant difference was found between the two parasite lines. Histopathological analysis revealed a greater inflammatory response in the placentas from NcGRA7KO-infected dams than in those from the parental strain. This finding correlates with upregulated chemokine mRNA expression for CCL2, CCL8, and CXCL9 in the placentas from the NcGRA7KO-infected mice. In conclusion, these results suggest that loss of NcGRA7 triggers an inflammatory response in the placenta, resulting in decreased vertical transmission of N. caninum.     

The last stronghold of Muñoa’s Pampas cat (Leopardus munoai) in Argentina?

The Muñoa’s Pampas cat (Leopardus munoai) is a relatively understudied species restricted to open savannas and grasslands of Southern Brazil, Uruguay and NE Argentina, that has been recently split from the broader “Pampas cat” species complex. Only three documented records of the species have been published since the year 2000 for the Argentine portion of its distribution, a situation that led to speculations regarding its conservation status in Argentina. We conducted an intensive camera-trap survey (2,067 camera-trapping stations and 15,560 camera-trapping days) to assess the presence of the Muñoa’s Pampas cat in an area of 275.3 km² within the Iberá National Park and the adjacent Iberá National Reserve, Corrientes province, Argentina. Four records of Muñoa’s Pampas cat were obtained, representing at least two adults and one young individual. Only one camera-trapping record of this species had been previously obtained in Argentina, during a survey carried out in 2009 in the same study area, despite an important camera-trapping effort in the Corrientes province. All camera-trap records of Muñoa’s Pampas cat in Argentina are concentrated in areas of temporally flooded grasslands locally known as “Malezales”, suggesting that this habitat type is critically important for the conservation of this rare felid. The Iberá National Park and the adjacent Iberá National Reserve provide the adequate framework for the conservation of an important piece of habitat for Muñoa’s Pampas cat and constitute a stronghold for the species in Argentina.     

Neospora caninum: Activity of cholinesterases during the acute and chronic phases of an experimental infection in gerbils

Neosporosis is an infectious disease primarily of dogs and cattle which has been found in many countries around the world. Neospora caninum causes an important immune response (cellular and humoral) in animals that it infects. Since the participation of the cholinergic system in the immune response is well documented, the aim of this study was to evaluate the relationship between N. caninum infection and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) during the acute and chronic phase of infection. For that, tachyzoites of N. caninum (Nc-1 strain) were inoculated intraperitoneally in gerbils (Meriones unguiculatus), which were separated in two experiments, I and II, differing in infective doses of tachyzoites, aiming to reach an acute phase as well as chronic phase, respectively. Samples were collected on day 7 post infection (PI) for Experiment I and on days 15 and 30 PI for Experiment II. AChE activity was evaluated on whole blood and brain, while BChE was evaluated in plasma. On day 7 a reduction of AChE in total blood and brain was observed, along with reduction of BChE in plasma of infected animals when compared with non-infected. In Experiment II, AChE activity increased in total blood on day 30 PI; however, maintaining, during the same period, the AChE activity has a reduced in brain tissue. BChE activity was significantly increased on day 30 PI. Based on the results obtained, it was possible to observe a response of the cholinergic system, providing different grades of AChE and BChE activities, in response to the acute and chronic infection of gerbils experimentally infected with N. caninum. These results will serve as initial points to further studies of our research group about the relationship between the infection/disease and the cholinergic system.     

Toxoplasma gondii and Neospora caninum infections in goat abortions from Argentina

The aims of this study were to identify the occurrence of Toxoplasma gondii and Neospora caninum abortions in goats from Argentina by serological, macroscopical and microscopical examination and bioassay, and to characterize the obtained isolates by molecular techniques. For this purpose, 25 caprine fetal fluids, 18 caprine fetal brains and 10 caprine placentas from 8 dairy/meat goat farms from Argentina were analyzed. Gestational age of the aborted fetuses was determined in 18 cases. Protozoal infections were detected by at least one of the applied diagnostic techniques in 44% (11/25) of examined fetuses; specifically, 24% (6/25) were positive to T. gondii, 8% (2/25) were positive to N. caninum and 12% (3/25) were positive to both parasites. In this study IFAT titers were similarly distributed in younger and older fetuses. Macroscopical and microscopical examination of one placenta revealed chalky nodules in the fetal cotyledons and normal intercotyledonary areas, as well as necrosis and calcification of mesenchymal cells in villi. Tachyzoites were observed in peritoneal wash from 2 mice inoculated with brain and a pool of brain and placenta of two fetuses. Cell culture growth of tachyzoites was achieved from one inoculated mouse, and confirmed as T. gondii by PCR. The T. gondii isolate was identified as atypical or non-canonical by nested-PCR-RFLP. This is the first study that investigated the involvement of N. caninum and T. gondii in cases of goat abortion in Argentina.     

Invasive alien plants in the Pampas grasslands: a tri-national cooperation challenge

The challenge of managing biological invasions requires novel approaches and coordinated efforts, especially among countries linked by intense trade routes and sharing common biomes. This is the case in Southern South America, where Argentina, Brazil and Uruguay maintain intense commercial relationships, whilst sharing continuous portions of the Pampas grasslands. Transnational similarities in this case exceed ecological features, including a common colonization history and similar development trends. This study represents a tri-national cooperation effort to describe the alien plant flora invading the Pampas grasslands of Argentina, Brazil and Uruguay and analyses their characteristics in order to detect species and traits that are shared among the three countries and those that have not yet managed to invade the whole region. Furthermore, we highlight the opportunities and needs of a common approach across countries to deal with plant invasions. Information about alien plant species was retrieved from the IABIN Invasives Information Network (I3N) project databases of Argentina, Brazil and Uruguay, complemented with some national herbaria. Three hundred and fifty-six alien plant species were recorded growing in natural or semi-natural habitats of the Pampas. A total of 50 species were found in Pampas grasslands of the three countries. Argentina shared 48 and 36 species with Brazil and Uruguay, respectively, while the Brazilian and the Uruguayan Pampas shared only 20 species. Poaceae, Asteraceae and Fabaceae were the families with the highest number of invasive species, and herbs were the most common life form (75 %). Most invasive plants originated from Europe, Asia and Africa, and almost one-quarter of the species is associated with some human use, especially gardening. We discuss the opportunities and needs for international cooperation, as the prevention of introductions from one country to another through the use of risk analysis tools, definition of priority invasive species, as well as the detection, containment, eradication and control of common invasions. Furthermore, we suggest the establishment of coherent regional legislation and the inclusion of social issues and the public perception in the biological invasion quest.     

First record of the freshwater snail Pseudosuccinea columella (Gastropoda: Lymnaeidae) in southern Pampas (Argentina) and assessment of future spread

ABSTRACT

The freshwater snail Pseudosuccinea columella was recorded for the first time in Argentina more than 60 years ago. Since then its distribution in the wild has been restricted to the northeastern provinces. Here we record the presence of P. columella in southern Pampas for the first time, extending its distribution more than 500?km southwards. The climatic suitability of this and other areas of South America for its establishment and spread was analysed using habitat modelling software. Hitherto its spread within and between watercourses in southern Pampas has been very limited, probably through a combination of low climatic suitability, recent introduction and low connectivity of the drainage basins. The suitability of other areas where it has been recently recorded indicates a moderate risk of further spread in central and northwestern Argentina and in coastal areas of the Pacific rim of South America (southern Perú and northern Chile). The recent spread of P. columella in the wild in Argentina may be the result of an increase in the trade in aquarium plants or of the evolution of a new lineage with different ecological capabilities.     

Morphological observation and molecular phylogeny of Spirometra decipiens complex 1 (Cestoda: Diphyllobothriidae) found in cat from Chile

The systematics of tapeworms in the genus Spirometra has been progressing with the accumulation of molecular genetics data, but the taxonomic status of many nominal species remains under debate. We report morphological and molecular-phylogenetic data for a Spirometra species collected from a domestic cat (Felis silvestris catus) in Chiloé Island, Chile. The Spirometra species was shown to be genetically conspecific with Spirometra decipiens complex 1 found in a Pampas fox (Lycalopex gymnocercus) from Argentina, and was closely related to a Hoary fox (Lycalopex vetulus) and rattlesnake (Crotalus durissus) from Brazil. Therefore, the presence of S. decipiens complex 1 was molecularly confirmed for the first time in Chile. The findings of the present study add useful information for the systematics of poorly known Spirometra species in South America.     

A novel real-time TaqMan™ PCR assay for simultaneous detection of Neotropical fox species using noninvasive samples based on <Emphasis Type="Italic">cytochrome c oxidase subunit II</Emphasis>

Strategies to evaluate and monitor elusive mammal species require the development of genetic techniques and their application to unambiguous biological material for ecological and genetic studies. In order to assess cytochrome c oxidase subunit II gene inter- and intraspecific variations, we compared sequences from different Neotropical canids and domestic dogs. We developed a primer pair to amplify a 154-bp fragment of this gene and a species-specific multiplex TaqMan? assay for accurate identification of two native fox species occurring in sympatry in South America, the crab-eating fox (Cerdocyon thous) and the pampas fox (Lycalopex gymnocercus). The assays can also distinguish domestic dogs (Canis lupus familiaris) from both wild foxes. The use of different fluorescent reporter dyes for species identification in a multiplex probe PCR-RT assay reduces labor and costs. The methodology presented in this study demonstrates an efficient approach to enable high-performance analysis and represents a reliable cost-effective tool for molecular ecology research to monitor the wild canid populations by noninvasive genetic sampling. This standardized assay will allow large-scale high-throughput analyses in a routine and reliable way.     

Neosporacaninum: Application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (M3-DPPE), referred to as M3-DPPE liposomes, have been shown to induce cellular immunity against antigens encapsulated therein. To evaluate whether these M3-DPPE liposomes have an adjuvant capacity against Neospora caninum infection, a novel immunization method utilizing soluble N. caninum apical membrane antigen 1 (NcAMA1) encapsulated in the M3-DPPE liposomes (M3-NcAMA1) was employed. The results revealed that a significant amount of interferon (IFN)-γ production was detected in culture supernatants of NcAMA1 protein- or N. caninum lysate-stimulated spleen cells obtained from the mice one week after the third immunization with M3-NcAMA1. The parasite burden in the dams’ brain tissue was decreased and the survival rate of offspring increased significantly in M3-NcAMA1-immunized mice. Thus, a parasite-specific Th1 immune response was successfully induced in the pregnant mice immunized with M3-NcAMA1, and an effective reduction of offspring mortality from N. caninum infection was triggered.     

Neospora caninum:Tachyzoites Express a Potent Type-I Nucleoside Triphosphate Hydrolase,but Lack Nucleoside Diphosphate Hydrolase Activity

Asai, T., Howe, D. K., Nakajima, K., Nozaki, T., Takeuchi, T., and Sibley, L. D.Neospora caninum: Tachyzoites Express Type-I Nucleoside Triphosphate Hydrolase1. But Lack Nucleoside Diphosphate Hydrolase Activity.Experimental Parasitology90,277–285. We have identified type I nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) activity, previously thought to be restricted to the virulent strains ofToxoplasma gondii, in the cell extracts ofNeospora caninumtachyzoites. Sequence analysis of a complete cDNA from Nc-1 strain indicated thatN. caninumNTPases shared approximately 69% identity to the NTPases ofT. gondiiand are most similar to the NTPase-I isozyme. Southern blot analysis of genomic DNA and sequence analysis of two independentNTPclones from the Nc-1 strain revealed the presence of multiple genes, at least two of which are transcribed. Substrate specificity andK_mvalues for MgATP²⁻and MgADP⁻hydrolysis for recombinant or partially purified native NcNTPase were the same as those for the type I isozyme (NTPase-I). Significantly, no type II enzyme (NTPase-II) activity for NDP hydrolysis was detected in cell extracts ofN. caninum, although it is universally present in allT. gondiistrains that have been tested. This intriguing difference between these two closely related apicomplexan parasites may provide insight into the function of the NTPases during intracellular parasitism.     

Status of the alien pathogen Paranosema locustae (Microsporidia) in grasshoppers (Orthoptera: Acridoidea) of the Argentine Pampas

After experimental introductions from North America in 1978–1982, the biocontrol agent Paranosema locustae became established in grasshopper communities of the western Pampas region of Argentina. The use and establishment of P. locustae in Argentina constitute both a case of neoclassical or new association biological control (use of an alien species against native pests) and a case of pathogen pollution (anthropogenic introduction–establishment of an infectious disease in populations of native species). Since P. locustae is a multihost pathogen among grasshoppers, its presence in the western Pampas represents an additional factor disrupting grasshopper communities according to the differential susceptibility of each species and possibly threatening some species. Microscopic examination of 504 grasshopper samples (the mean number of individuals per sample = 185) belonging to 43 species from 93 localities throughout the Pampas revealed an establishment area of approximate 90,000 km² from about 35° North to 38° South and from 61° East to 65° West. Field infections by P. locustae have now been detected in 21 grasshopper species in the western Pampas. Susceptible species with geographic distributions mostly restricted to the establishment area and with numerically small populations, like the melanopline Scotussa daguerrei, are predicted to be the ones facing higher risks of negative impacts.     

Role of exotic tree stands on the current distribution and social behaviour of Swainson's hawk, Buteo swainsoni in the Argentine Pampas

Aim The Argentine Pampas was extensively and abruptly altered by European colonization. Between 1880 and 1885 the indigenous human inhabitants were completely displaced, and native grasslands were replaced by exotic pasture plants and crops. One of the most important ecological changes in the Pampas landscape, the introduction of tall exotic tree species, has received little attention, and its effect on wildlife has never been assessed. We have made an intensive survey of habitat use of Swainson's hawk, Buteo swainsoni in its most important non‐breeding quarters, the Pampas of Argentina, aiming to characterize the sites used by hawks for communal roosting. Location Pampas grasslands, Argentina. Methods We surveyed 30,000 km of roads by car during the austral summer from 2001 to 2004, covering the main non‐breeding area occupied by Swainson's hawks. Their roost sites were located by direct observation of birds roosting in tree stands close to the roads, by surveying potential roost sites around places where groups of pre‐ and post‐roosting hawks were recorded, and by gathering information from local farmers. Results Swainson's hawks exclusively used stands of exotic tree species for roosting. Eucalyptus viminalis was present in all 34 roosts surveyed, and in 59% of them it was the only species present. The remaining exotic tree species were the Siberian elm, Ulmus pumila, pines (Pinus spp.) and cypress (Cupressus spp.). Flock sizes at roost sites were unusually high for a raptor, with an average of 658 individuals (range 8–5000 hawks, n = 27 flocks). Main conclusions The introduction of exotic trees may have resulted in the expansion of the suitable habitat for Swainson's hawks, permitting a recent colonization of the Argentine Pampas. Tree stands may have also changed the communal roosting behaviour of this raptor, by virtue of their providing new structural elements in a region that almost completely lacked trees prior to European occupation.     

Dietary Spectrum of Two Sympatric Canid Species in Ladakh,India

We have conducted a study between May and August 2013 to understand the dietary spectrum of two canid species: Tibetan wolf (Canis lupus chanco) and Red fox (Vulpes vulpes) in one of the most inhospitable landscape of the world. Ladakh is characterized by harsh environmental conditions and low primary productivity and therefore animal populations, particularly of large and medium sized herbivores are also low and sparsely distributed. This in turn poses a challenge to the survival of predators. We have attempted to investigate the ways by which the two sympatric canid species are optimizing feeding for their survival in an environment where there is scarcity of wild prey base. Scats of the two species were collected from Chiktan and Rangdum valleys and undigested food remnants were identified. The dietary spectrum of Tibetan wolf was composed of six prey species (2 wild and 4 livestock species). The share of livestock species in the diet was maximum (RO: 62.0%). The Red fox being an opportunistic feeder consumed 19 forgeable items, including small and medium sized mammals, birds, reptiles, insects, plant material, eggs, stones and debris. The livestock species contributed maximum to the diet of Red fox (RO: 22.9%). The dietary niche breadths of both species were more or less similar (0.53 and 0.58) and indicating a generalist feeding behavior. Both the species are surviving in the same environment with a dietary overlap of 0.60. The given information is expected to aid further studies in Indian Trans-Himalaya.     

Australian dingoes are definitive hosts of Neospora caninum

To provide objective data on the potential role of dingoes (Canis lupus dingo) in the life cycle of Neospora caninum in Australia, the production of N. caninum oocysts by experimentally infected canids was investigated. Three dingo pups raised in captivity and three domestic dogs were fed tissue from calves infected with an Australian isolate of N. caninum, Nc-Nowra. Oocysts of N. caninum, confirmed by species-specific PCR, were shed in low numbers by one dingo pup at 12-14 days p.i. The remaining animals did not shed oocysts. Furthermore, the blood from two out of three dingoes tested positive for DNA of N. caninum using PCR tests at 14 and 28 days p.i. Oocyst shedding from the intestinal tract of a dingo demonstrates that dingoes are definitive hosts of N. caninum and horizontal transmission of N. caninum from dingoes to farm animals and wildlife may occur in Australia.     

Learning interspecific communicative responses in Pampas foxes (Lycalopex gymnocercus)

Domestic dogs show remarkable communicative abilities in their interaction with people. These skills maybe explained by the interaction between the domestication process and learning experiences during ontogeny. Studies carried out on other species of canids, which have not been domesticated are relevant to this topic. The purpose of this article is to study the effect of instrumental learning on captive Pampas foxes’ (Lycalopex gymnocercus) communicative responses to humans. Seven foxes were tested in a conflict situation involving food within sight but out of their reach. In these situations dogs typically gaze at the human face to ask for food. In Study 1, there was an increase in gaze duration as a consequence of reinforcement and a decrease during extinction, when animals did not receive any more food. In Study 2, all of the four foxes tested successfully followed proximal pointing gesture to find hidden food. When a distal pointing cue was given, three out of four followed it in the first session and one in the second session. These results are consistent with those previously found in dogs, and suggest that learning experiences allow the development of communicative skills, even in not domesticated canid species.