Role of wild rabbits as reservoirs of leishmaniasis in a non-epidemic Mediterranean hot spot in Spain.

There is limited information regarding the role of wild mammals in the transmission dynamics of Leishmania infantum. A potential human leishmaniasis hot spot was detected in southern Spain that could not be explained solely by canine leishmaniasis prevalence. The aim of this work was to analyse the involvement of wild rabbits as the main factor affecting this Mediterranean hot spot. A survey of wild rabbits, dogs and sand flies was conducted in the human cases environment. A nearby region without clinical leishmaniasis cases was used as reference control. 51 wild rabbits shot by hunters were analysed by molecular techniques. 1100 sand flies were captured and morphologically identified. Blood collected from patients' relatives/ neighbours (n = 9) and dogs (n = 66) was used for molecular analysis and serology. In Mediterranean leishmaniasis hot spots such as Montefrío municipality (average incidence of 16.8 human cases per 100,000 inhabitants/year), wild rabbits (n = 40) support high L. infantum infection rates (100%) and heavy parasite burdens (average value: 503 parasites/mg) in apparently normal ear skin directly accessible to sand flies, enabling the existence of heavily parasitized Phlebotomus perniciosus females (12.5% prevalence). The prevalence of infection and median parasite load were very low among rabbits captured in Huéscar (n = 11), a human clinical leishmaniasis-free area for the last 18 years. P. perniciosus was the most abundant Phlebotomus species in all the domestic/peridomestic microhabitats sampled, both indoors and outdoors. Accordingly, leishmaniasis is clustering in space and time at this local scale represented by Montefrío due to the proximity of two competent host reservoirs (dogs and heavily parasitized wild rabbits) associated with overlapping sylvatic and domestic transmission cycles through the main vector, P. perniciosus. We highlight the usefulness of determining the prevalence of infection and parasite burden in wild rabbits as a control leishmaniasis measure with the advantage that the use of the ear offers.

Evaluation on two types of paramyosin vaccines for the control of Haemaphysalis longicornis infestations in rabbits.

BACKGROUND: Haemaphysalis longicornis is an obligate hematophagous ectoparasite that transmits a variety of pathogens causing life-threatening diseases in humans and animals. Paramyosin (Pmy) is not only an invertebrate-specific myofibrillar protein but also an important immunomodulatory protein. Therefore, it is one of the ideal candidate antigens for vaccines. METHODS: We conducted two vaccine trials to evaluate the protective efficacy of Pmy recombinant protein (rPmy) and peptide vaccine (KLH-LEE). Each rabbit was immunized with three doses of rPmy or KLH-LEE adjuvanted with Freund's complete/incomplete at 500 µg/dose at 2-week intervals before challenge with 40 female H. longicornis/rabbit. PBS plus adjuvant, Trx or KLH was used as control group. The antibodies of rabbits were detected by ELISA. Then, female ticks were fed on the rabbits until detachment. RESULTS: ELISA results showed that both vaccines induced rabbits to produce antibodies. Compared with the Trx group, the engorgement weight, oviposition and hatchability of the rPmy group decreased by 8.87%, 26.83% and 38.86%, respectively. On the other hand, engorgement weight, oviposition and hatchability of female ticks in the KLH-LEE group correspondingly resulted in 27.03%, 53.15% and 38.40% reduction compared with that of the KLH group. Considering the cumulative effect of vaccination on the evaluated parameters, results showed 60.37% efficacy of the rPmy vaccine formulation and 70.86% efficacy in the KLH-LEE group. CONCLUSIONS: Pmy and particularly epitope LEE have potential for further development of an effective candidate vaccine to protect the host against tick infection. GRAPHIC ABSTARCT.

Global transcriptome landscape of the rabbit protozoan parasite Eimeria stiedae.

BACKGROUND: Coccidiosis caused by Eimeria stiedae is a widespread and economically significant disease of rabbits. The lack of studies on the life-cycle development and host interactions of E. stiedae at the molecular level has hampered our understanding of its pathogenesis. METHODS: In this study, we present a comprehensive transcriptome landscape of E. stiedae to illustrate its dynamic development from unsporulated oocysts to sporulated oocysts, merozoites, and gametocytes, and to identify genes related to parasite-host interactions during parasitism using combined PacBio single-molecule real-time and Illumina RNA sequencing followed by bioinformatics analysis and qRT-PCR validation. RESULTS: In total, 12,582 non-redundant full-length transcripts were generated with an average length of 1808 bp from the life-cycle stages of E. stiedae. Pairwise comparisons between stages revealed 8775 differentially expressed genes (DEGs) showing highly significant description changes, which compiled a snapshot of the mechanisms underlining asexual and sexual biology of E. stiedae including oocyst sporulation between unsporulated and sporulated oocysts; merozoite replication between sporulated oocysts and merozoites; and gametophyte development and gamete generation between merozoites and gametocytes. Further, 248 DEGs were grouped into nine series clusters and five groups by expression patterns, and showed that parasite-host interaction-related genes predominated in merozoites and gametocytes and were mostly involved in steroid biosynthesis and lipid metabolism and carboxylic acid. Additionally, co-expression analyses identified genes associated with development and host invasion in unsporulated and sporulated oocysts and immune interactions during gametocyte parasitism. CONCLUSIONS: This is the first study, to our knowledge, to use the global transcriptome profiles to decipher molecular changes across the E. stiedae life cycle, and these results not only provide important information for the molecular characterization of E. stiedae, but also offer valuable resources to study other apicomplexan parasites with veterinary and public significance.

Seasonal Dynamics of Fleas (Siphonaptera: Pulicidae, Ceratophyllidae and Leptopsyllidae) on Oryctolagus cuniculus in a Meso-Mediterranean Area of Central Spain.

Flea infestations of wild rabbits were examined monthly in central Spain in a meso-Mediterranean area for 5 yr. A total of 1,180 wild rabbits were trapped and 7,022 fleas were collected from them. Overall, the prevalence was 74.1% with a mean flea index of 5.95 fleas per rabbit. Four flea species were identified: Spilopsyllus cuniculi (Dale, 1878) was the most abundant species (accounting for 74.3% of fleas collected) followed by Xenopsylla cunicularis (Smit, 1957), Odontopsyllus quirosi (Gil Collado, 1934), and Nosopsyllus fasciatus (Bosc, 1800) (18.9, 6.7, and 0.1%, respectively). The highest prevalence was observed in S. cuniculi (48.6%) followed by X. cunicularis, O. quirosi, and N. fasciatus (34.3, 20.0, and 0.6%, respectively). Odontopsyllus quirosi and S. cuniculi were mainly collected from autumn to spring with the peak of infestation in winter, while X. cunicularis was mainly found from spring to autumn with maximum levels of infestation during the summer months. The relevance of these findings is discussed.

Rabbit Bot Fly Furuncular, Tracheopulmonary, and Human Bot Fly Infestations in Connecticut (Oestridae: Cuterebrinae).

Endemic and tropical human bot infestations are relatively uncommon or unreported in the United States. We report two cases in Connecticut: an unusual furuncular and respiratory myiasis by the rabbit bot Cuterebra buccata (Fab.) (Diptera: Oestridae) in a 74-yr-old male and a case of human bot fly, Dermatobia hominis (L.) (Diptera: Oestridae), myiasis in a 4-yr-old female with a tropical travel history with her family. Identification of C. buccata was based morphologically, in part, on spinal armature and further corroborated by DNA sequencing of the mitochondrial COI gene and comparison to the National Center for Biotechnology Information GenBank DNA sequence database. The resulting annotated sequence data were deposited into the National Center for Biotechnology Information GenBank. The unique medical aspects, and limitations and specifics on bot fly larval habits and identification are discussed.

Surveillance of Zoonotic Parasites in Animals Involved in Animal-Assisted Interventions (AAIs).

Animal-assisted interventions (AAIs) are based on the establishment of a therapeutic relationship between animals and beneficiaries that is certain to provide positive effects, while currently, it reads as if AAIs aim at exposing stakeholders to potential risk of infection. The surveillance of zoonotic pathogens is necessary for guaranteeing common health. This study investigated the presence of potentially zoonotic parasites, including dermatophytes, in animals involved in AAIs. Between 2015 and 2017, 190 animals (equids, dogs, cats, birds, rabbits, rodents, and goats) were investigated. Anamnestic and management data were recorded. Individual faecal samples were analysed using a copromicroscopic procedure. Fur and skin were examined for ectoparasites during clinical examinations, and samples for mycological investigation were collected by brushing. Parasites were described in 60 (31.6%) investigated animals. Thirteen out of the 60 (21.7%) animals harboured potentially zoonotic parasites, mainly recovered in dogs (Ancylostomatidae, Eucoleusaerophilus, Toxocaracanis, and Giardiaduodenalis) and a cat (G.duodenalis). Nannizziagypsea and Paraphytonmirabile, potential agents of cutaneous mycosis, were isolated in a dog and a horse, respectively. No ectoparasites were found. AAIs might represent a source of infections either directly or via environmental contamination. Thus, active surveillance is necessary and animal screenings should be planned and scheduled according to the risk of exposure.

Presence of eimerid oocysts in faeces of a quarantined dog in Iceland is explained by coprophagic behaviour prior to its importation. Case report.

BACKGROUND: All dogs imported into Iceland must undergo mandatory quarantine in a special station before introduction into the country. A faecal sample is collected from the first stool passed by the dog in this station and subsequently examined for the presence of intestinal parasite stages. CASE PRESENTATION: In May 2019 unsporulated oocysts were detected in faeces from a 7-year-old household dog that had been imported from Sweden. Most of the oocysts studied strongly resembled those of Eimeria canis Wenyon, 1923. As this species is not valid, the purpose of the present article was to identify the correct species and examine their possible origin. Studies confirmed the presence of two distinct unsporulated oocyst morphotypes in the faeces; measurements and photomicrographs confirmed their identification as Eimeria magna Pérard, 1925 and Eimeria stiedai (Lindemann, 1865) Kisskalt and Hartmann, 1907, both common parasites of European rabbits, Oryctolagus cuniculus (L., 1758). When the owner of the dog was questioned about the food administrated to the dog prior to its import to Iceland, it turned out that it had exclusively been fed dry dog food pellets. However, the owner also reported that on the morning prior to transportation to Iceland, the dog was allowed to move freely in a grassland area where rabbits are common and heaps of their faeces are present. Furthermore, the owner confirmed that the dog consumed rabbit faeces that morning. CONCLUSION: It is believed that this coprophagic behaviour can explain the detection of rabbit eimerids in the dog's faeces, and that such behaviour must be taken into consideration by veterinarians and other diagnostic personnel when they detect atypical cysts or eggs during coprological examinations.

New record of Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) from Brazil with an illustrated key to species for the genus.

The cheyletid mites that parasitize mammals have been neglected for a long time in Brazil, although they can be common on pets and cause injury to their hosts. Recently, Cheyletiella parasitivorax was found parasitizing a rabbit in Brazil which represents a new host and distribution record for the mite species. An illustrated dichotomous key for the identification of the species in this genus and data from the literature are provided.

Small animal in vivo imaging of parasitic infections: A systematic review.

Non-invasive small animal in vivo imaging is an essential tool in a broad variety of biomedical sciences and enables continuous monitoring of disease progression in order to develop and improve diagnostic, therapeutic and preventive measures. Imaging parasites non-invasively in live animals allows efficient parasite distribution evaluation in the host organism and objective evaluation of parasitic diseases' burden and progression in individual animals. The aim of this systematic review was to summarize recent trends in small animal in vivo imaging and compare and discuss imaging of single-cell and multicellular eukaryotic parasites. A literature survey was performed using Web of Science and PubMed databases in research articles published between 1990 and 2018. The inclusion criteria were using any imaging method to visualize a range of protozoan and helminth parasites in laboratory animals in vivo. A total of 92 studies met our inclusion criteria. Protozoans and helminths were imaged in 88% and 12% of 92 studies, respectively. The most common parasite genus studied was the protozoan Plasmodium followed by Trypanosoma and Leishmania. The most frequent imaging method was bioluminescence. Among the helminths, Schistosoma and Echinococcus were the most studied organisms. In vivo imaging is applicable in both protozoans and helminths. In helminths, however, the use of in vivo imaging methods is limited to some extent. Imaging parasites in small animal models is a powerful tool in preclinical research aiming to develop novel therapeutic and preventive strategies for parasitic diseases of interest both in human and veterinary medicine.

Sarcocystis Infection in Laboratory Rabbits.

Sarcocystosis, presumably caused by Sarcocystis cuniculi, was diagnosed in 2 purpose-bred, SPF Dutch belted laboratory rabbits from a class A breeder. The rabbits were purchased by a research facility and conventionally housed individually in stainless-steel suspended caging. At necropsy and tissue harvest, gross lesions were not observed in the muscles. Upon histologic examination, sarcocysts were found in the eyelid of one rabbit and the tongue of the other. To our knowledge, this report is the first description of infection by Sarcocystis spp. in laboratory rabbits.

First Report of Obeliscoides cuniculi in the European Rabbit (Oryctolagus cuniculus).

PURPOSE: Obeliscoides cuniculi is a common parasite in American lagomorphs, while it has never been reported in the European rabbit. The exotic parasite has been introduced in Italy by the eastern cottontail (Sylvilagus floridanus), an invasive alien lagomorph introduced to Europe for hunting purpose. Considering that S. floridanus shares the same areas with the European rabbit, the aim of this work is to document the first report of this parasite in an autochtonous lagomorph. METHODS: O.cuniculi, isolated from one adult European rabbit found dead, was identified considering the morphological features and reproductive organs of the adult stage (length and thickness of the body, spicules structure and size). RESULTS: The present study represents the first record of the gastric nematode O. cuniculi (Trichostrongilidae) in a population of European rabbit (Oryctolagus cuniculus) in Italy. CONCLUSION: Since Graphidium strigosum is the most common parasite infecting the European rabbit stomach, this study considers and discusses different scenarios about helminth co-infection effect. Further studies should be carried out to understand the long-term effect of this introduction on wild rabbit population.

Molecular characterization of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Eimeria spp. and Blastocystis are the common parasites that parasitize the intestinal tract of rabbits, which can seriously threaten the health of rabbits and lead to economic losses to the rabbit industry. However, information about the prevalence and transmission of these two parasites in rabbits is limited in China. The objective of this study was to survey the prevalence of Eimeria spp. and Blastocystis in rabbits in Shandong Province. A total of 616 rabbit fecal samples were collected from two cities (Rizhao and Weihai) in Shandong Province, eastern China, and Eimeria spp. and Blastocystis were identified by polymerase chain reaction based on species-specific markers. The prevalence of Eimeria spp. was 20% (123/616) and the Blastocystis prevalence was 0.97% (6/616). Five different Eimeria species (Eimeria intestinalis, E. perforans, E. magna, E. media, and E. irresidua) and the ST4 subtype of Blastocystis were identified in rabbits by sequence analysis. This is the first report of Blastocystis prevalence and subtype ST4 in rabbits in Shandong Province. The findings provide baseline data for the prevention and control of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Coccidiosis ­ a problem in backyard rabbitries

This paper focuses on parasites of the genus Eimeria in rabbits kept in two backyard farms ­ from Poland (herd A) and Ukraine (herd B). The concentration McMaster technique and sporulation was applied to determine the level of infection, as well as to identify the species composition. The Eimeria sp. oocysts were present in all collected samples (prevalence = 100%), and four to nine coccidian species were identified in the herd A and B, respectively. Eimeria media was the most common species, and the presence of the most pathogenic E. intestinalis and E. flavescens was observed in both studied rabbitries. The results obtained suggest that coccidiosis may cause a serious risk for rabbits kept in backyard farms, therefore prevention guidelines in relation to the backyard breeding are crucial to be developed.

Prevalence and Genotype Distribution of Giardia duodenalis in Rabbits in Shandong Province, Eastern China.

Giardia duodenalis is a zoonotic enteric parasite that can infect humans and a number of animal species including rabbits with a worldwide distribution. Infection with G. duodenalis can cause serious public health problems and significant economic losses to animal husbandry. So accurate understanding of the prevalence and genotype distribution of G. duodenalis in rabbits is necessary. In the present study, a total of 616 fecal samples were collected from rabbits in Shandong province, eastern China, and examined in G. duodenalis prevalence and genotypes by nested PCR amplification of ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) gene loci of G. duodenalis. Sixty-nine (11.2%) of the examined rabbit fecal samples were G. duodenalis-positive. Of them, the prevalence of G. duodenalis is 8.4% (41/490) in Rizhao city and 22.2% (28/126) in Weihai city. Breeds, region, and feeding modes were highly correlated with G. duodenalis infection in rabbits. Moreover, three genotypes (assemblages A, B, and E) were identified in rabbits at three gene loci, and the assemblage E was the dominant genotype, while the assemblage A was reported in rabbits in China for the first time. It is noticeable that two rabbits were found to be infected with two different G. duodenalis assemblages (assemblages A and E, assemblages B and E, respectively). These findings enrich the genotype distribution of G. duodenalis in rabbits and provide baseline data for preventing and controlling G. duodenalis infection in rabbits in eastern China.

Comparison of Seroprevalence and PCR Results in the Detection of Toxoplasma gondii in Pet Rabbits in Poland.

Toxoplasma gondii infections are prevalent in humans and animals worldwide. The aim of the study was to estimate the seroprevalence of T. gondii in pet rabbits, as well the presence of T. gondii DNA in their blood. A total of 360 pet rabbits were investigated for the presence of antibodies and antigens of T. gondii in blood samples using a modified agglutination test (MAT) and nested PCR, respectively. Antibodies against T. gondii were found in 44 (12.12%) of pet rabbits. In rabbits that received unwashed vegetables 13.13% were positive for T. gondii antibodies, whereas all rabbits that received washed vegetables were seronegative. The prevalence of anti-T. gondii antibodies was statistically higher in samples collected from ill rabbits (45.45%) compared with healthy ones (8.87%), as well in the group of rabbits that had contact with cats (16.13%) compared with those without contact with cats (3.57%). The percentage of seropositive samples derived from all three sampling regions of Poland was as follows: Silesia (12.20%), Lower Silesia (6.09%), and Lesser Poland (18.03%). No statistical differences in seroprevalence were observed according to age or sex of rabbits. All tested blood samples were negative for the T. gondii B1 gene in nested PCR. This is the first study of seroprevalence and the presence of T. gondii in the blood of pet rabbits in Poland. Our study indicates that health status of rabbits, contact with cats, as well sampling region may have an important impact on the prevalence of T. gondii infection.

New record of Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) from Brazil with an illustrated key to species for the genus / Novo registro de Cheyletiella parasitivorax (Mégnin, 1878) (Trombidiformes: Cheyletidae) para o Brasil, com uma chave ilustrada das espécies deste gênero

Abstract The cheyletid mites that parasitize mammals have been neglected for a long time in Brazil, although they can be common on pets and cause injury to their hosts. Recently, Cheyletiella parasitivorax was found parasitizing a rabbit in Brazil which represents a new host and distribution record for the mite species. An illustrated dichotomous key for the identification of the species in this genus and data from the literature are provided.

Resumo Os ácaros da família Cheyletidae que parasitam mamíferos são negligenciados há muito tempo no Brasil, embora eles sejam comuns em animais domésticos. Considerando as dificuldades morfológicas para diagnosticar as espécies dessa família que infestam mamíferos, este estudo refere-se a uma nova ocorrência de Cheyletiella parasitivorax incluindo os poucos registros de literatura. Além disso, também está sendo apresentada uma chave dicotômica ilustrada para identificação de espécies desse gênero.

Immune protection provided by a precocious line trivalent vaccine against rabbit Eimeria.

A pilot study was conducted to evaluate the pathogenicity and immunogenicity of vaccinated rabbits with different doses of oocysts (5 × 102, 1 × 103, 1 × 104, and 5 × 104) of a precocious line, including Eimeria magna, E. intestinalis or E. media following the challenge with their corresponding parent strains. Our results showed that each precocious line had weak pathogenicity but good immunogenicity in terms of clinical symptoms, average daily weight gains (ADGs), and oocyst outputs. Therefore, a precocious line trivalent vaccine, including E. magna, E. intestinalis, and E. media was formulated. A total of sixty 40-day-old coccidia-free rabbits were allocated to ten treatments with a 2 × 3 factorial arrangement that included 2 vaccination doses (5 × 102 or 1 × 103 oocysts of the precocious line). Groups I to Ⅷ and Unimmunized Challenged Control group were challenged with mixed oocysts of their corresponding parent strains (1 × 104 oocysts of each parent strain) 14 days after vaccination. No clinical symptoms were observed in the immunized groups after vaccination. Average daily weight gains (ADGs) were similar to those of unimmunized unchallenged controls (P > 0.05) after vaccination or after challenge. Oocyst outputs in the vaccinated challenged groups were significantly different from those of unimmunized challenged controls (P < 0.01) after challenge. These results indicated that the trivalent vaccine could provide immune protection against coccidiosis and therefore, it could be used as a candidate vaccine.

A pathological study of Leishmania infantum natural infection in European rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis).

In this study, we describe the pathology of Leishmania infantum infection in naturally infected wild Leporidae and compare diagnosis of infection using histopathology, direct fluorescent antibody (DFA) assay, immunofluorescence antibody test (IFAT) and quantitative real-time PCR (qPCR). Tissues were analysed from 52 European rabbits (Oryctolagus cuniculus) and 7 Iberian hares (Lepus granatensis) from the Community of Madrid (Spain). Our results show that L. infantum infection is associated with only minimal histopathological lesions and that L. infantum amastigotes can be detected by DFA assay in all tissues types tested, including skin. These results were confirmed by qPCR on fresh frozen tissues in 13% of rabbits and 100% of hares. However, L. infantum DNA could not be detected by qPCR on paraffin-embedded tissue obtained by laser capture microdissection. Using the DFA assay to diagnose L. infantum, infection may provide further insights into this disease in wild animals and may allow the precise tissue localization of L. infantum, thereby guiding follow-up tests with more accurate qPCR.

Gastrointestinal parasites of the New England cottontail rabbit (Sylvilagus transitionalis) and eastern cottontail rabbit (Sylvilagus floridanus) in the Hudson Valley, New York.

The New England cottontail rabbit (NEC, Sylvilagus transitionalis) population has decreased dramatically in New York, USA, and the role of parasites in limiting the population has never been examined. The closely related and sympatric eastern cottontail rabbit (EC, Sylvilagus floridanus) was introduced into the range of NEC by humans and is currently thriving. This study aimed to investigate gastrointestinal parasites of the NEC and the EC and compare their parasite communities. Fecal pellets from 195 NEC and 125 EC were collected from the Hudson Valley, New York, in the winter of 2013-2014. Centrifugal fecal floats were performed in Sheather's sugar solution, and parasite ova and cysts were examined microscopically to identify gastrointestinal parasites present. For all pellets combined (n = 320), 91% were found to harbor at least 1 parasite species, with Eimeria species being the most common. Genetic analysis of pellets using microsatellite DNA identified 248 individual rabbits, with parasite prevalence (94%) similar to the prevalence estimate based on all pellets (91%). EC samples had a significantly higher (p < 0.05) parasite species richness (1.73, range 0-4) than NEC (1.20, range 0-3). EC and NEC shared 3 moderate to high (9-89%) prevalence parasites, in which EC prevalence was consistently higher. One parasite species was only found in NEC, and two were only found in EC, but the majority of these were of low abundance, precluding further statistical analyses.

Occurrence and genetic characterization of Toxoplasma gondii and Neospora caninum in slaughtered domestic rabbits in central China.

Currently, information on the occurrence and genetic characterization of Toxoplasma gondii and Neospora caninum in tissues of rabbits in China is lacking. In this study, brain and heart samples from 470 slaughtered domestic rabbits were collected in Henan Province, Central China. The occurrence rate of T. gondii and N. caninum DNA detected by nested PCR was 2.8% and 2.1%, respectively. There were no significant differences (p > 0.05) in the frequency of the two parasite infections in relation to sex, breed, and region. Three out of 13 T. gondii-positive samples were completely or partially genotyped at 11 genetic markers using PCR-RFLP, and one was identified as ToxoDB genotype #9. For N. caninum, three different sequences at the ITS1 region and two genotypes at the MS5 microsatellite locus were identified. To our knowledge, this is the first genetic characterization of N. caninum isolates from rabbits.