Cryogenic preservation of Dirofilaria immitis microfilariae, reactivation and completion of the life-cycle in the mosquito and vertebrate hosts.

BACKGROUND: The cryopreservation of filarial nematodes has been studied for nearly 70 years. Largely, these studies examined the effectiveness of cryopreservation methods by using the post-thaw survival of microfilariae (mf) and the development to third-stage larvae (L3s) following inoculation into a competent insect vector. Only one study reported complete reestablishment of a filarial nematode (Brugia malayi) life-cycle in a competent vertebrate host from cryopreserved stock. Expanding on this previous research, a cryopreservation method was developed to cryopreserve the mf of the dog heartworm, Dirofilaria immitis. METHODS: A combination of cryoprotectants, dimethyl sulfoxide (DMSO) and polyvinyl pyrolidone (PVP) at 6% and 4 mM, respectively, provided acceptable post-thaw survival of mf that developed into L3s in Aedes aegypti. L3s developed from cryopreserved and freshly collected mf in mosquitoes were inoculated into ferrets and dogs and were assessed after a sufficient duration post-inoculation for development into adult heartworms. RESULTS: Fewer adult heartworms derived from cryopreserved stocks of mf were recovered from ferrets compared to adult heartworms derived from freshly collected mf, and the former were smaller by weight and length. The onset of patency (circulating mf) occurred at similar post-inoculation time points and at similar mf densities in dogs infected with L3s sourced from cryopreserved stocks or freshly collected mf. Adults derived from cryopreserved mf have survived and produced viable mf for more than 3 years in dogs. Approximately 60% of inoculated L3s were recovered as adults from dogs at 2 and 3.5 years post-inoculation. CONCLUSIONS: The results from these direct comparisons demonstrate that cryopreserved mf can develop into L3s in vector mosquitoes and that these L3s are infective to both dogs and ferrets, where they undergo normal development into adult worms. These worms are able to mate and produce viable mf and complete the heartworm lifecycle in dog.

First record of Troglotrema acutum (Trematoda, Troglotrematidae) in European polecats Mustela putorius from Luxembourg.

We report the first record of the trematode Troglotrema acutum in skulls of European polecats (Mustela putorius) from Luxembourg. Thirty-one road-killed polecats from the northern and eastern parts of the country were examined. We found the trematode in three polecats from the administrative districts of Clervaux and Vianden in the northeast of the country. The parasites were detected in the frontal sinuses of one juvenile male as well as one adult of each sex. The animals were infected with six, nine and 13 adult trematodes, respectively, and we recovered 24 trematode eggs from one polecat. While the two adult hosts showed the lesions and deformations of the skull that are typical of T. acutum infections in polecats, the skull of the juvenile was not deformed. The prevalence of 9.7% reported here was lower than the values reported from Central European studies. Our findings indicate that the distribution range of T. acutum in western Central Europe extends to the Ardennes low mountain range.

Antibodies to Leishmania in naturally exposed domestic ferrets (Mustela putorius furo) in Spain.

Zoonotic leishmaniosis due to Leishmania infantum is a vector-borne disease endemic in southern Europe and dogs are the main reservoir for this infection. Seasonal variations in antibody titers in this species in areas where phlebotomine vectors have seasonal patterns of activity are important for epidemiological, preventive and clinical studies related with canine leishmaniosis. It has been suggested that cats, rabbits and ferrets may act as peridomestic reservoirs and not only as accidental hosts. The aim of this study was to determine if seropositive ferrets (Mustela putorius furo) to Leishmania could be affected by seasonal variations of anti-Leishmania antibodies. A group of seropositive clinically healthy ferrets (n = 21) were included in this study. A significant reduction in anti-Leishmania infantum antibodies was detected during non-transmission period (December 2020-February 2021) in comparison to transmission period (April-October 2020). This study describes for the first time a seasonal variation in the anti-Leishmania antibodies detected in domestic ferrets following natural exposure during sand fly transmission period and the following non-sand fly transmission period in a Mediterranean area considered as an area where L. infantum is endemic.

Molecular characterization of enteric coccidia from domestic ferrets (Mustela putorius furo).

Combined morphometric and molecular characterization of coccidia that infect domestic ferrets (Mustela putorius furo) was completed to improve the diagnostic specificity of 'coccidiosis' in this host. Coccidia-positive fecal samples (n = 11) and formalin-fixed paraffin-embedded intestinal tissues (n = 3) from domestic ferrets were collected from diagnostic laboratories in Canada and Europe. An average of 3.5 and 13 domestic ferret fecal samples per year were coccidia-positive when tested by Canadian and European diagnostic laboratories, respectively, during the period 2008-2015. Oocyst morphometrics and sequence genotyping at two loci (nuclear 18S rDNA [nu 18S] and mitochondrial cytochrome c oxidase subunit I [mt COI]) were conducted on all samples. The first nu 18S and mt COI sequences for Cystoisospora laidlawi, and the first mt COI sequence for Eimeria furonis were generated during this study. Phylogenetic analysis of the mitochondrial COI sequences demonstrated that E. furonis was most closely related to E. ictidea isolated from a black-footed ferret (Mustela nigripes) and that C. laidlawi was closely related to C. canis and C. felis. The identifications provided by diagnostic laboratories of the specific parasite species present in a sample showed poor agreement with their identifications based on genotyping obtained in this study. Molecular techniques appear to be essential for determining the specific coccidial species responsible for individual and group outbreaks of coccidiosis and for further understanding of eimeriid host-parasite relationships.

ENCEPHALITOZOON CUNICULI INFECTION IN A BLACK-FOOTED FERRET (MUSTELA NIGRIPES) KIT.

A 47-day-old black-footed ferret (Mustela nigripes) kit was found dead in June 2016. Histologic examination revealed pyogranulomatous tubulointerstitial nephritis, pneumonia, and encephalitis, with intralesional microsporidia. Transmission electron microscopic examination showed microsporidia with ultrastructural characteristics consistent with Encephalitozoon spp. Polymerase chain reaction (PCR) and direct sequencing confirmed the presence of Encephalitozoon cuniculi genotype II. This organism has been reported in other Carnivora (i.e., canids, felids, mustelids, procyonids, otariids). In humans, it is generally described as an opportunistic pathogen in immunocompromised individuals. The source of infection in the quarantine facility remains unknown, although two groups of frozen feeder rat kidneys tested positive for E. cuniculi genotype II via PCR. Feeding whole prey to various zoo taxa carries some potential disease transmission risk.

Tick-borne pathogens in the European polecat, Mustela putorius and in attached Ixodes hexagonus ticks from Germany.

The European polecat, Mustela putorius, occurs almost throughout Europe. However, there is a lack of data on the ectoparasite fauna and the potential role in the circulation of tick-borne pathogens (TBP) of this mustelid species. The aim of this study was to investigate whether M. putorius contributes to the maintenance of TBP in Germany. DNA samples extracted from spleen tissue of 117 M. putorius, which had been collected mainly in North-western Germany from 2012 to 2015, were tested by real-time and conventional PCRs for Anaplasma phagocytophilum, Babesia spp., Bartonella spp., Candidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. In addition, 200 randomly selected engorged Ixodes hexagonus ticks (100 females and 100 nymphs) collected from 39 of the 88 M. putorius were tested for these TBPs, except for Hepatozoon spp., and additionally for Borrelia spp. and Rickettsia spp. Three of six pathogens were detected in the spleen tissue of the 117 M. putorius: A. phagocytophilum (n = 5; 4.3%), Babesia cf. odocoilei (n = 1; 0.9%) and CNM (n = 1; 0.9%), including one case of co-infection (A. phagocytophilum and CNM). Ixodes hexagonus ticks tested positive only for Bartonella spp. (26/200 ticks; 13.0%) which were detected exclusively in adult female ticks. Sequencing revealed the presence of Bartonella taylorii and uncultured Bartonella spp. The results suggest that M. putorius neither seems to serve as the main reservoir nor plays an important role in maintaining TBPs in Germany but may rather contribute to the dilution of these pathogens. However, M. putorius contributes to the maintenance of tick populations, especially of I. hexagonus. The high prevalence of Bartonella spp. in I. hexagonus ticks may suggest a certain importance of this tick species in the maintenance of these bacteria in nature.

Molecular identification of Sarcocystis lutrae (Apicomplexa: Sarcocystidae) in muscles of five species of the family Mustelidae.

Carnivores usually act as definitive hosts of Sarcocystis species. However, the number of reports on sarcocyst formation in musculature of predators is on the increase. In the present study, muscle samples of 68 mustelids collected in Lithuania were examined for sarcocysts of Sarcocystis species. Sarcocysts were detected in diaphragm, tongue and limb muscles of ten animals (14.7%) but were not discovered in the heart. Based on 18S rDNA, 28S rDNA, cox1 and ITS1 sequence analysis, Sarcocystis lutrae was identified in three American minks (Neovison vison), two beech martens (Martes foina), three Eurasian badgers (Meles meles), one Eurasian otter (Lutra lutra) and one European polecat (Mustela putorius). The intraspecific variability of this Sarcocystis species was determined only in ITS1 region. Based on the phylogenetic analysis, no clear separation of S. lutrae by intermediate hosts or geographical locations was established. This paper represents the first identification of S. lutrae in the American mink, the beech marten and the European polecat. Current results indicate that S. lutrae is a common species in the muscles of various European mustelids.

Brugia malayi infection in ferrets - A small mammal model of lymphatic filariasis.

BACKGROUND: The lack of effective short-course therapies for treatment of the adult stage of filarial worms is a major limitation in the global effort to eliminate lymphatic filariasis. Studies using current small mammal models of lymphatic filariasis are limited by difficulties in quantifying adult worm numbers and in assessing lymphatic anatomy and function. METHODOLOGY/PRINCIPAL FINDINGS: Here, we re-established Brugia malayi infection of ferrets as a model for lymphatic filariasis and demonstrated parasitological, immunological, and histological parallels with human infection. Subcutaneous injection of L3 larvae into a hind-footpad resulted in a mean of 18 adult worms recovered 16 weeks post-infection, primarily from the draining inguinal and femoral lymphatics of the injected limb. Infected ferrets developed microfilaremia, with patency lasting from 12-26 weeks post-infection. Quantitative PCR assessing cytokine transcription by antigen-stimulated lymph node cells demonstrated a mixed Th1/Th2 response occurring during early infection. Immunoregulation with production of down-regulatory cytokine IL-10 occurred just prior to peak microfilaremia. Histological analysis revealed progressive inflammation of the lymphatic vessel walls, with intimal thickening and disorganization of collagen fibers. Inflammation was observed as early as 8 weeks post-infection and extended into the perivascular and subcutaneous tissues by 16 weeks post-infection. Finally, we developed a novel ferret PET/CT lymphoscintigraphy method demonstrating substantial changes in lymphatic anatomy and function as early as 3 weeks post-infection, with progression over the course of infection. CONCLUSIONS/SIGNIFICANCE: B. malayi infection of ferrets is a robust model of human lymphatic filariasis that can be utilized to study efficacy of novel antifilarial agents against adult worms residing within lymphatic vessels. In conjunction with PET/CT lymphoscintigraphy, this model can also be used to investigate pathogenesis of lymphatic dysfunction in lymphatic filariasis and efficacy of medications aimed at reversing lymphatic dysfunction after clearance of adult worms.

Filarial dermatitis caused by Filaria taxideae in domestic ferrets (Mustela putorius furo) from the western United States.

Filaria taxideae is a common subcutaneous filarial parasite of American badgers (Taxidea taxus) that has also been reported in skunks in the Western USA. Dermatitis associated with this parasite has been reported in badgers and skunks; however, many individuals have no gross lesions. With the exception of a captive red panda in California, there have been no reports of F. taxidae infection in domestic or exotic/pet animals. In this case series, we document F. taxideae in domestic ferrets from California, Texas, and Colorado and describe the clinical presentation, gross and histologic lesions, and the molecular characterization of this parasite. In two cases, ferrets were positive on Dirofilaria immitis antigen tests but had no evidence of heartworm infection suggesting that F. taxideae antigens can cross-react. These findings indicate that F. taxideae should be considered in cases of dermatitis in ferrets in areas where this parasite may occur.

Possible Role of Fish as Transport Hosts for Dracunculus spp. Larvae.

To inform Dracunculus medinensis (Guinea worm) eradication efforts, we evaluated the role of fish as transport hosts for Dracunculus worms. Ferrets fed fish that had ingested infected copepods became infected, highlighting the importance of recommendations to cook fish, bury entrails, and prevent dogs from consuming raw fish and entrails.

FLEAS OF BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) AND THEIR POTENTIAL ROLE IN THE MOVEMENT OF PLAGUE.

Sylvatic plague is one of the major impediments to the recovery of the black-footed ferret ( Mustela nigripes ) because it decimates their primary prey species, prairie dogs ( Cynomys spp.), and directly causes mortality in ferrets. Fleas are the primary vector of Yersinia pestis , the causative agent of sylvatic plague. The goal of this research was to better understand the flea fauna of ferrets and the factors that might influence flea abundance on ferrets. Fleas from ferrets were tested for Y. pestis in a post hoc assessment to investigate the plausibility that some ferrets could act as incidental transporter hosts of fleas infected with Y. pestis . Fleas were collected from ferrets captured on the Lower Brule Indian Reservation in central South Dakota, US from 2009 to 2012. A total of 528 fleas collected from 67 individual ferrets were identified and tested for the presence of Y. pestis with a nested PCR assay. The predominant flea recovered from ferrets was Oropsylla hirsuta , a species that comprises 70-100% of the fleas recovered from prairie dogs and their burrows in the study area. Yersinia pestis was detected at low levels in fleas collected from ferrets with prevalence ranging from 0% to 2.9%; male ferrets harbored significantly more fleas than female ferrets. Six of 67 ferrets vaccinated against plague carried fleas that tested positive for Y. pestis , which suggests ferrets vaccinated against plague could inadvertently act as incidental transporter hosts of Y. pestis -positive fleas.

First survey of endoparasites in pet ferrets in Italy.

Endoparasites are infrequently reported in ferrets. A cross-sectional survey was conducted to determine the prevalence of intestinal parasites in pet ferrets in southern Italy. Fresh fecal samples were randomly collected from 50 ferrets housed in pet shops or privately owned. All fecal samples were processed using the FLOTAC pellet technique to identify and count helminthic eggs/larvae and protozoan cysts/oocysts. In addition, the samples were analyzed also by the Remel XpectGiardia/Cryptosporidium immunoassay. Intestinal parasites were detected in 15 out of 50 ferrets (30%). Eggs of ancylostomids were found in 28.0% (14/50) of the animals and oocysts of Sarcocystis were detected in one ferret (2.0%). None of the samples was positive for Cryptosporidium or Giardia. To the authors' knowledge, this is the first report of sarcosporidiosis in a pet ferret in Italy.

Occurrence and molecular typing of Giardia isolates in pet rabbits, chinchillas, guinea pigs and ferrets collected in Europe during 2006-2012.

A total of 1180 faecal samples (528 from rabbits, 531 from chinchillas and 121 from guinea pigs) collected during 2006-2012 by veterinarians in Germany and in other European countries were submitted to a diagnostic laboratory for Giardia testing by means of coproantigen ELISA. Of these samples, 40 rabbits (7.6 per cent), 326 chinchillas (61.4 per cent) and five guinea pigs (4.1 per cent ) were found to be positive. To gain insights into the genetic identity of Giardia in small mammals, ELISA-positive samples from 23 chinchillas, five ferrets, a rabbit, and a Desmarest's hutia were investigated by PCR and sequencing of fragments of the small subunit ribosomal DNA (ssu), the triose phosphate isomerase (tpi) and the ß-giardin (bg) genes. At the ssu locus, assemblage B was identified in 28 of 30 isolates, whereas assemblage A and D were each detected in one sample. The majority of isolates from chinchillas and those from ferrets had Giardia duodenalis sequences identical to sub-assemblages AI or BIV, based on either a single locus (tpi or bg) or multiple loci (tpi and bg). As sub-assemblages AI or BIV are associated with human infection, these results indicate that small mammals can act as reservoirs of cysts potentially infectious to humans.

Ectoparasites in black-footed ferrets (Mustela nigripes) from the largest reintroduced population of the Conata Basin, South Dakota, USA.

The black-footed ferret, Mustela nigripes, is an endangered carnivore endemic to the grasslands of North America. We present the first investigation of ectoparasites associated with black-footed ferrets since reintroduction. We sampled more than 200 individuals from one of the largest and most successful reintroduced populations located in the Conata Basin of South Dakota, USA. We compared our findings with ectoparasite assemblages of sympatric carnivores and historic ferret records. We collected more than 1,000 ectoparasites consisting mainly of three flea and tick species, two of which were known historically from South Dakota. Despite our extensive sampling efforts, we did not detect any lice. This is notable because a putative host-specific louse, Neotrichodectes sp., was presumed to have gone extinct when black-footed ferrets were extirpated from the wild. The ectoparasite assemblage on black-footed ferrets comprised only generalist parasites, particularly those found on their prey such as prairie dogs (Cynomys sp.). Oropsylla hirsuta was the most abundant ectoparasite, representing 57% of all ectoparasites detected; a flea vector important in the persistence and transmission of plague. Black-footed ferrets like other endangered species undergo repeated parasite removal and vaccination efforts to facilitate population recovery, which may have unintentionally contributed to their depauperate ectoparasite community.

Outbreaks of severe enteric disease associated with Eimeria furonis infection in ferrets (Mustela putorius furo) of 3 densely populated groups.

CASE DESCRIPTION: 3 unrelated, densely populated, dynamic ferret populations with severe outbreaks of enteric coccidiosis were evaluated. CLINICAL FINDINGS: In each outbreak, morbidity rate was high, there were an appreciable number of deaths, and ferrets of all ages were affected. Affected individuals had acute onset of diarrhea, and feces often contained frank or digested blood. Other clinical signs included dehydration, weakness, lethargy, and weight loss. Fecal examinations of affected ferrets revealed sporadic and inconsistent shedding of coccidial oocysts. Necropsy findings included moderate to marked atrophic enteritis associated with numerous intraepithelial and fewer extracellular coccidial life stages. Sporulated oocysts isolated from feces were consistent with Eimeria furonis. A PCR assay was performed on formalin-fixed, paraffin-embedded sections of intestine for the gene encoding the small subunit of rRNA yielded products with sequences identical to those described for E furonis. TREATMENT AND OUTCOME: Supportive care and treatment with sulfadimethoxine over the course of these outbreaks was palliative, but long-term treatment was required and failed to completely eradicate infection as identified by the subsequent finding of oocysts in fecal samples. CLINICAL RELEVANCE: Enteric coccidiosis due to infection with E furonis has typically been reported to be subclinical rather than to cause severe gastrointestinal disease in ferrets. This report indicated that infection with E furonis may have contributed to severe enteric disease with high morbidity and mortality rates in 3 densely populated, dynamic groups of ferrets. Furthermore, long-term treatment with anti-coccidials may be required in outbreak situations, but may be ineffectual in completely eradicating infection.

Efficacy and safety of the combination imidacloprid 10 % / moxidectin 1.0 % spot-on (Advocate(®) spot-on for small cats and ferrets) in the treatment of ear mite infection (Otodectes cynotis) in ferrets.

In this study, the efficacy and safety of a treatment with the combination imidacloprid 10 %/ moxidectin 1.0 % spot-on (Advocate(®) spot-on for small cats and ferrets) was tested in 39 ferrets naturally infested with ear mites (Otodectes cynotis). The study was performed as a multicentre, non-randomised, non-controlled (all study animals were treated) and non-blinded clinical field study in two French veterinary practices. Four visits (day (D) 0 = inclusion and first treatment, D14 = second treatment, D28 = possible third treatment, D56 = termination) were planned. The dosage was one pipet per ferret (designed for cats weighing up to 4 kg, corresponding to a dose of moxidectin ranging from 2.2 to 5 mg/kg body weight) two or three times at 14-days intervals (at D0, D14 and possibly D28 depending on the parasitological examination of the ears at D28). The main efficacy criterion was the absence of the parasite (all stages incl. eggs, larvae, nymphs and adults) from ear scrapings by microscopic examination. At D28 after two treatments (D0 and D14), 76.9 % (30/39) of animals were cured. Only 23 % (9/39) needed a third treatment. At day 56, 100 % were cured. Local symptoms (inflammation and pruritus) were consistently improved (50.6 % improvement at D14, 81.0 % at D28 and 97.9 % at D56) as well as the abnormal cerumen production (14.7 % improvement at D14, 77.7 % at D28 and 100.0 % at D56). No general symptoms were noticed during the study (general health and skin aspect). Advocate(®) spot-on for small cats and ferrets is an effective and safe treatment for ear mite infection in ferrets. Two or three treatments administered in 14-days intervals to ferrets infested with ear mites provided 100 % parasitological cure on D56.

Autochthonous Dirofilaria immitis infection in a ferret with aberrant larval migration in Europe.

A two-year-old male ferret (Mustela putorius furo) was presented to the Faculty of Veterinary Science, Szent István University, for investigation of somnolence. Following unsuccessful therapeutic attempts, the ferret was euthanased and a male Dirofilaria immitis worm was found in the pulmonary artery and a female D. immitis specimen in the subdural space of the cranial cavity. To the authors' knowledge, this is the first European record of D. immitis infection in a ferret, and the first case in which aberrant larval migration and consequent central nervous system signs were observed in a ferret in the course of D. immitis infection.

Disseminated, histologically confirmed Cryptococcus spp infection in a domestic ferret.

CASE DESCRIPTION: A 4-year-old castrated male domestic ferret from central Massachusetts was evaluated for weight loss over a 1.5-month period and for 2 days of retching, diarrhea, and signs of lethargy. It had been housed indoors, with 2 other ferrets, 2 cats, and humans that lacked signs or symptoms of disease. CLINICAL FINDINGS: Physical examination revealed a thin body condition, tachypnea, an increase in respiratory effort, and retching. Splenomegaly was detected during abdominal palpation. Clinicopathologic analysis revealed lymphopenia, lactic acidosis, hypoglycemia, hypocalcemia, hypoalbuminemia, and hyperglobulinemia. A pulmonary bronchointerstitial pattern was evident on radiographs, and abdominal ultrasonography revealed a suspected pancreatic mass and mesenteric lymphadenopathy. TREATMENT AND OUTCOME: After 2 weeks of medical treatment and once clinical signs resolved, an exploratory laparotomy was performed and a lymph node biopsy specimen was collected. Histologic evaluation of the specimen revealed Cryptococcus-like organisms. Antifungal treatment was initiated with itraconazole (PO) and amphotericin B (IV). The ferret died after 2 days of treatment. A full necropsy was performed, revealing multicentric cryptococcosis affecting the lungs, brain, spleen, and multiple lymph nodes. Paraffin-embedded, formalin-fixed lung tissue was submitted for DNA extraction, and the organism was identified as Cryptococcus neoformans var grubii. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of disseminated cryptococcosis in a North American ferret. This case is unique in that the ferret lived indoors, in a geographic region in which reports of cryptococcosis are rare. The genotyping technique used to identify the Cryptococcus strain can aid in better understanding the epidemiology of cryptococcosis.

Transvenous heartworm extraction in a ferret with caval syndrome.

A 10-month-old ferret was diagnosed with heartworm disease and caval syndrome. Associated clinical signs included weakness and a green-colored urine, identified as biliverdinuria. Despite the animal's small size, removal of three heartworms via transvenous heartworm extraction was successfully performed. Although at least one female worm remained in the right ventricle, the majority of clinical signs related to the presence of the heartworms resolved. The ferret was subsequently managed medically with corticosteroids and monthly heartworm prevention. This case documents the presence of biliverdinuria associated with caval syndrome and successful transvenous heartworm extraction in a ferret.