Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.     

Prevalence of dirofilarial infection in raccoon dogs in Japan.

The raccoon dog (Nyctereutes procyonoides viverrinus) is known to acquire canine heartworm (Dirofilaria immitis) infection. We surveyed the prevalence of heartworm infection in free-ranging raccoon dogs in the Nishi-Tama (Tokyo) and Kanagawa areas of Japan. A total of 75 raccoon dog carcasses, including 29 animals from the Nishi-Tama area and 46 from the Kanagawa area, were necropsied between 1992 and 1993. Eight out of 75 raccoon dogs were found to be infected (overall 10.7%). The prevalence of infection was 6 and 16% in Nishi-Tama and Kanagawa, respectively. Microfilarial production was observed in the uterus of one female adult dog.     

Experimental infection of Paragonimus iloktsuenensis to albino rats, dogs and cats

This study was performed to observe the susceptibility of dogs and cats as definitive hosts of Paragonimus iloktsuenensis. The metacercariae of this fluke were obtained from Sesarma dehaani collected at a focus near the mouth of Sumjin river in November, 1986 and February, 1987. The larvae isolated from the crabs were introduced per os into 7 albino rats, 2 dogs and 3 cats. The adults were recovered from the experimental animals, and they were morphologically observed and measured. The results were as follows: 1. The recovery rate of adult worms at 42 days after infection was 53.3% from three albino rats, 21.0% from a dog and 12.7% from two cats. Most of the worms were recovered from the worm capsules in the lungs. 2. The size of worms recovered from albino rats, a dog, and cats 42 days after infection averaged 6.3 x 3.2 mm, 6.3 x 3.0 mm, or 6.2 x 3.5 mm, respectively. There were little differences in the morphology of worms by different experimental animals. 3. The size of eggs from a dog was 88.9 x 49.3 microns, and that from cats was 84.3 x 53.7 microns on average. Dogs and cats were good definitive hosts of P. iloktsuenensis. This fact suggests that human infection by this fluke may be possible if the metacercariae were ingested.     

Detection of Echinococcus granulosus coproantigens in Australian canids with natural or experimental infection

Coproparasitological and purging methods for diagnosing canids infected with the intestinal helminth Echinococcus granulosus, an important zoonotic parasite, are unreliable. Detection of coproantigens in feces of infected dogs by enzyme-linked immunosorbent assay (ELISA) is suitable for detecting patent and prepatent infections with a high degree of sensitivity and specificity. In the present study, natural and experimental infections in domestic and wild Australian canids were investigated using a coproantigen capture ELISA. Experimental infection of dogs with E. granulosus was detected at between 14 and 22 days postinfection (PI), and optical density (OD) values remained high until termination of experiments 35 days PI. After chemotherapy, coproantigen levels in infected dogs dropped rapidly, becoming negative 2-4 days after treatment. In experimentally infected red foxes (Vulpes vulpes), the coproantigen excretion profile was different, with ELISA OD levels peaking 15-17 days PI, then falling to low or undetectable levels by 30 days PI. Coproantigens were detected in the feces of naturally infected Australian wild dogs (dingoes, dingo/domestic dog hybrids) with infection levels ranging between 2 worms and 42,600. Preliminary data on the stability of coproantigen in dog feces exposed to environmental conditions indicated that there was no change in antigenicity over 6 days. The results suggest the coproantigen ELISA could be successfully used to monitor E. granulosus prevalence rates in Australian domestic dogs, foxes, and wild dogs.     

Active and passive immunization of mice against larval Dirofilaria immitis

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.     

Laboratory observations on the insusceptibility of raccoons to Dirofilaria immitis

Two raccoons, Procyon lotor, were exposed to Dirofilaria immitis by subcutaneous injection of infective third stage larvae obtained from experimentally-infected Aedes trivittatus. Nematodes were not recovered from either raccoon when examined at necropsy 223 and 254 days postexposure. Large numbers of adult D. immitis were found in six dogs used as controls. These data indicate that raccoons cannot support the development of D. immitis.     

Dirofilaria immitis: experimental infections in the ferret (Mustela putorius furo)

The ferret, Mustela putorius furo, was found to be susceptible to Dirofilaria immitis infection when exposed to low (14) or high (280-420) numbers of infective larvae harvested from Aedes aegypti. Eight ferrets (half of them cortisonized) were inoculated subcutaneously with 14 larvae each. All of them were subsequently found to harbor D. immitis in the heart, and all but one of them had worms of both sexes. Six of these ferrets were examined for microfilaremia at 31 to 35 weeks after inoculation; 3 were positive (one observed only at postmortem examination) and there was evidence that fertilization of female worms had occurred in one other. Females up to 25.5 cm and males up to 16.0 cm were recovered. There was no evidence that the cortisonization of some ferrets had affected the infections. Both male and female ferrets became infected. Four cortisonized ferrets were inoculated with 280 or 420 larvae of D. immitis (divided equally between subcutaneous and intraperitoneal routes). All of them died 16 to 18 weeks after inoculation, yielding 102 to 125 immature D. immitis. In these lethal infections, worms were recovered from the heart and adjoining vessels, and also from vascular and extravascular sites throughout the body.     

Susceptibility of avian hosts to experimental Gymnophalloides seoi infection

To determine whether avian species are susceptible to infection with Gymnophalloides seoi (a human-infecting intestinal trematode), we exposed 7 species of birds with metacercariae obtained from oysters. The birds were necropsied at days 2, 4, and 6 postinfection (PI). The highest worm recovery at day 6 PI was obtained from the Kentish plover (Charadrius alexandrinus; mean = 56.0%), followed by the Mongolian plover (C. mongolus; 49.3%), and the grey plover (Pluvialis squatarola; 32.3%). In contrast, no mature worms were recovered from the great knot (Calidris tenuirostris), dunlin (C. alpina), black-tailed gull (Larus crassirostris), and mallard (Anas platyrhynchos). Among the plovers, the worms attained the greatest size at day 6 PI (254.1 x 190.4 microm) in the Kentish plover, with a significantly higher number of eggs in the uterus. The 3 species of plovers are highly susceptible to experimental G. seoi infection, suggesting that they could play a role as definitive hosts for these worms in nature.     

Dirofilaria immitis: identification and partial characterization of parasite antigens in the serum of infected dogs

We have recently developed a sensitive and specific immunodiagnostic test for canine Dirofilaria immitis infection based on detection of soluble parasite antigens in dog sera by monoclonal antibody-based enzyme immunoassay. In addition to their importance as markers of infection, these antigens may contribute to the pathogenesis of heartworm disease in dogs. In the present study, a variety of methods were used to identify and characterize circulating D. immitis antigens. Two antigens were identified in infected dog sera that formed lines of identity in rocket-line immunoelectrophoresis with soluble antigens extracted from adult D. immitis. Circulating D. immitis antigens were also demonstrated in infected dog sera by immunoblot analysis with polyclonal and monoclonal antibodies. These antigens had apparent molecular weights that ranged from 50 to 250 kDa. Most of the circulating D. immitis antigens contained the epitope defined by monoclonal antibody 1418BF2.1 which is used in our enzyme immunoassay for circulating D. immitis antigen. Studies of parasite antigens released during in vitro culture indicated that the circulating D. immitis antigens in dog sera that are detected by our enzyme immunoassay are primarily derived from adult female worms.     

Serological and molecular studies on Dirofilaria immitis in dogs from Turkey

We estimated the prevalence of Dirofilaria immitis infection in domestic dogs in five Turkish provinces - Sakarya, Kocaeli, Ankara, Elazig and Mersin - using a commercial ELISA kit for detecting circulating antigen and a PCR test for detecting circulating microfilarial DNA. A total of 211 whole-blood and serum samples were collected from dogs of various breeds, ages and life status (owned or stray). Sample population characteristics were recorded and examined for differences in prevalence. Additionally, we collected 15 blood samples from cats (14 owned and 1 stray) from Ankara province and used PCR to detect D. immitis infection. Twenty-seven (12.8%) of 211 dog samples were positive for D. immitis antigen by ELISA. No differences in prevalence were observed by sex (female: 14.4%; male: 10.7%; P>0.05). The prevalence of D. immitis infection varied with age: 11.8% in younger dogs (0.5-2 years) and 17.5% in older dogs (3-5 years). Prevalence between stray dogs (15.2%) and owned dogs (9.3%) did not differ (P>0.05). Prevalence rates were highest in Kocaeli province (18.3%), followed by Ankara (14.8%), Sakarya (12.3%) and Mersin (10.5%) provinces. Prevalence in Elazig province was 0%. No dogs or cats had microfilarial DNA detectable by PCR.     

Canine heartworm in the domestic and wild canids of southeastern Nebraska

The prevalence of canine heartworm (Dirofilaria immitis) was examined in the domestic dog, coyote (Canis latrans), and red fox (Vulpes fulva) populations of southeastern Nebraska. Microfilariae were detected in 21.4% (22 of 103) of the domestic dogs. The average age of infection for dogs was 5.8 yr. Nine of the 22 infected dogs also were positive for Dipetalonema reconditum. Thirty-nine of 443 (8.9%) coyotes were found to have adult heartworms. The average number of male and female worms per heart was 3.7 and 3.9, respectively. The mean age of infected coyotes was 3.6 yr. Red foxes had an infection rate of 4.8% (1 of 21). This fox heart had only 1 immature female worm. A mail survey of 6 veterinary clinics was also conducted. Veterinarians reported infection rates of 0% to 18.8% in domestic dogs for their localities.     

The first record of Dirofilaria immitis infection in a Humboldt penguin, Spheniscus humboldti

Dirofilaria immitis infection is an important parastic disease in many mammals, especially canids, but has not been reported in bird hosts. Filarial worms were isolated from the lumen of the right atrium of the heart and the connective tissue of the lung of a captive female Humboldt penguin, Spheniscus humboldti, that died at a zoo in Japan. One of these worms was observed morphologically and identified as D. immitis by features such as 4 pairs of cephalic papillae, 1 pair of cervical papillae, esophagus divided into 2 regions, 4 pairs of pre-anal papillae, 5 pairs of post-anal papillae, and unequal spicules. In addition, the partial DNA sequence (234 bp) of mitochondrial CO / gene of the filarial worm was identical to that of D. immitis. This is the first report of D. immitis infection in a bird.     

Heterologous transmission with Dictyocaulus capreolus from roe deer (Capreolus capreolus) to cattle (Bos taurus)

Eight Swedish Red Breed cattle, about 2 months old, were experimentally infected with a Swedish isolate of Dictyocaulus viviparus (Dviv-Se) from cattle and D. capreolus from roe deer. The aims were to determine whether the roe deer lungworm is infective to cattle or if it can induce seroconversion in cattle against D. viviparus as measured with an ELISA. Four calves which were given 500 Dviv-Se infective larvae (L3) each by larval dosing for two successive days developed patent infection between days 23 and 25 post-inoculation (PI). Larval output varied among the calves and during the patent period. However, maximum recovery occurred between 28 and 56 days PI with peak shedding on day 37 PI. Shedding ceased at day 58 PI and adult worms were recovered from one calf at necropsy (day 67 PI). No immature worms were recovered from the lungs at necropsy. Seroconversion was detected on days 35-42 PI. One Dviv-Se infected calf became seronegative on day 67 PI whereas the other calves still remained seropositive during this period. Prepatency and patency periods of D. viviparus and serological findings in this study basically conform to previous studies. Each calf that was infected with 400 L3 of D. capreolus for two successive days, and about 800 L3 of the same species about 8 weeks later, did not develop to patency based on faecal and post-mortem examinations. Consequently, under the conditions of this study, D. capreolus was not infective to cattle. Two of the four calves that were infected with L3 from roe deer were challenged with L3 cultured from faeces of the Dviv-Se-infected calves. This infection did not develop to patency. Whether this was due to cross-protection as a result of the prior priming with L3 from roe deer is not clear. However, if it is so, it opens up the possibility of using D. capreolus L3 for preventing bovine dictyocauliasis.     

Immunisation of mice with fractions derived from the intestines of Dirofilaria immitis

Antigens that are not normally seen by the host but that are nevertheless, accessible to host immune effector molecules and cells such as the native endoantigens associated with the intestinal epithelium of haematophagous tissue-dwelling parasites, could be potentially useful vaccine antigens. In this study, intestines were dissected from adult Dirofilaria immitis, homogenised, and a 105,000 x g pellet obtained and extracted with Triton X-100. The soluble 105,000 x g supernatant from this extract induced partial protection (51%) against a challenge infection of third stage larvae (L3) implanted in micropore chambers. Sera from mice immunised with this soluble detergent extract reacted with proteins ranging in size from 38 to 130 kDa. Immunolocalisation studies indicated the mouse sera reacted primarily to the lumenal surface of the intestines of adult D. immitis, though reactivity to the lateral nerve/epithelial chords, hypodermis and reproductive tracts was also noted, indicating the presence of shared antigens. Tissues of L3s were also recognised by the immunised mouse sera. These mouse sera did not react to a dog blood fraction prepared identically to the D. immitis fraction. Only those sera from D. immitis-infected dogs with heavy or long-term infections were reactive to a single 42 kDa protein. After 24 h incubation in fluorescein isothiocyanate-conjugated serum the intestinal tract of Onchocerca volvulus and D. immitis L3 and L4 fluoresced, indicating the serum had been ingested. These data suggest that filarial gut-associated antigens (apart from the single 42 kDa antigen) are not seen by normally infected hosts, that they can be accessible to antibodies and that they can induce an immune response which is partially protective.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

Passive transfer of protective immunity to larval Dirofilaria immitis from dogs to BALB/c mice

Protective immunity to larval Dirofilaria immitis has been demonstrated in both the natural host, the dog, and in an experimental host, the mouse. In the present study, sera were collected and pooled from dogs that had been shown to have protective immunity to larval D. immitis. The pooled serum was inoculated into normal BALB/cByJ mice that then were challenged with third-stage larvae (L3) implanted in diffusion chambers. Two weeks postchallenge no significant difference was seen in either parasite survival or growth. Three weeks postchallenge, there was a significant decrease in parasite survival in mice receiving serum from immune dogs. Living larvae recovered at 3 wk postchallenge were significantly shorter than cohorts recovered from control mice. Antibody responses to L3 and forth-stage larvae (L4) surface antigens, to L3 and L4 aqueous soluble antigens, and to an excretory-secretory antigen fraction were measured. Only antibody responses to L3 surface antigens were elevated in the immune serum as compared to controls, thus suggesting a possible role for antibodies with specificity for surface antigens in protective immunity.     

Prevalence of canine dirofilariasis in Taiwan

Between 1993 and 1997, 837 stray dogs from North Taiwan were necropsied and examined for dog heartworm infections. A thick smear from 20 ml of peripheral blood from each dog was also prepared and examined for microfilariae (mf). The overall prevalence of adult worms in the dog population was 57%. The prevalence of mf in 1228 house dogs from different parts of Taiwan was also determined from 20 ml of peripheral blood in the same way. The overall prevalence of mf was 25%, with a value of 30% in the main island of Taiwan, this being 15 times higher than that in the offshore islands (2%). In Taiwan, the prevalence ranged from 4% in Hualien County, East Taiwan, to 41% in Nantou County, Central Taiwan. The mf prevalences on offshore islands were 1% on Liuchiu and 2% on Lanyu. The mf density per 20 ml blood in 82 house dogs was found to be 23 mf per dog, with a range of 3-97 mf per dog. A total of 477 stray dogs were found to be infected with adult worms of Dirofilaria immitis. The mean number of 7 worms per dog was obtained, with a range of 1-55 worms per dog. These results indicate that the prevalence of canine dirofilariasis has increased in Taiwan over the past 10 years. Moreover, the prevalence may be related to the wind speed, temperature, relative humidity, and altitude in the different areas surveyed.     

Survey of canine heartworm in the city of Recife, Pernambuco, Brazil.

Six hundred and eleven random-source dogs (338 male, 273 female) one year of age or older, from six sections of the city of Recife, Pernambuco, were examined antemortem for circulating microfilariae Dirofilaria immitis and Dipetalonema reconditum adult heartworm (D. immitis) antigen, and examined postmortem for adult heartworms. The prevalence of heartworm infection was 2.3% (14/611), as determined by necropsy for adult worms, and 1% (6/611) had circulating microfilariae of D. immitis; thus, 57.1% of the heartworm-infected dogs had occult infections. The results of serological testing indicated that 1.3% (8/611) of the dogs were positive for adult heartworm antigen. A total of 42 (6.9%) of the dogs had microfilariae of D. reconditum; 40 of these had only D. reconditum and two additional dogs had microfilariae of both species, D. immitis and D. reconditum.     

中国貉随机扩增多态DNA及其亚种分化关系

对来自陕西、云南、越南、安徽和广西等地的８只中国貉（Ｎｙｃｔｅｒｅｕｔｅｓｐｒｏｃｙｏｎｉｄｅｓ）进行随机扩增多态ＤＮＡ分析。应用２８个１０ｂｐ的随机引物,平均每只貉获得的ＲＡＰＤ标记数约为１３０条。遗传距离计算结果显示,中国貉个体间的平均遗传距离指数值为１１．２０％,最大值为１４．９３％,最小值为２．９４％。以赤狐（Ｖｕｌｐｅｓｖｕｌｐｅｓ）为外群,应用ＰＨＹＬＩＰ３．０计算软件包中的ＵＰＧＭＡ和ＮＪ聚类方法构建分子系统树。结果表明,不同地理群体间的中国貉存在遗传分化;中国貉可分为４组：（１）广西貉,（２）安徽貉,（３）陕西貉,（４）云南貉和越南貉。其中安徽貉和广西貉间的关系稍近,陕西貉则与云南貉－越南貉稍近。对合中国貉的形态分类、地理分布、ｍｔＤＮＡ多态分析以及进化遗传学的观点,认为陕西貉、广西貉和安徽貉可能与云南貉－越南貉具有等同的分类地位。