Antibody Detection and Molecular Characterization of Toxoplasma gondii from Bobcats (Lynx rufus), Domestic Cats (Felis catus), and Wildlife from Minnesota,USA

Little is known of the epidemiology of toxoplasmosis in Minnesota. Here, we evaluated Toxoplasma gondii infection in 50 wild bobcats (Lynx rufus) and 75 other animals on/near 10 cattle farms. Antibodies to T. gondii were assayed in serum samples or tissue fluids by the modified agglutination test (MAT, cut‐off 1:25). Twenty nine of 50 bobcats and 15 of 41 wildlife trapped on the vicinity of 10 farms and nine of 16 adult domestic cats (Felis catus) and six of 14 domestic dogs resident on farms were seropositive. Toxoplasma gondii oocysts were not found in feces of any felid. Tissues of all seropositive wild animals trapped on the farm were bioassayed in mice and viable T. gondii was isolated from two badgers (Taxidea taxus), two raccoons (Procyon lotor), one coyote (Canis latrans), and one opossum (Didelphis virginiana). All six T. gondii isolates were further propagated in cell culture. Multi‐locus PCR‐RFLP genotyping using 10 markers (SAG1, SAG2 (5′‐3′SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22‐8, c29‐2, L358, PK1, and Apico), and DNA from cell culture derived tachyzoites revealed three genotypes; #5 ToxoDataBase (1 coyote, 1 raccoon), #1 (1 badger, 1 raccoon, 1 opossum), and #2 (1 badger). This is the first report of T. gondii prevalence in domestic cats and in bobcats from Minnesota, and the first isolation of viable T. gondii from badger.     

Seroprevalence of antibodies to Toxoplasma gondii in the Pennsylvania bobcat (Lynx rufus rufus)

From 2000 to 2002 bobcat blood samples were collected, in association with the Pennsylvania Game Commission, during the recently reactivated bobcat hunting and trapping season. Sex, age, and county/township data were recorded for each animal. Blood was tested for antibodies to Toxoplasma gondii using the modified agglutination test. In the 2-yr study, 131 bobcat samples were collected in 14 Pennsylvania counties and 109 (83%) of these had antibodies to T. gondii (titer>or=25). A two-way Chi-Square test (95% confidence interval) yielded no significance differences in antibody prevalence between males (83%) and females (88%) or adults (83%) and juveniles (77%). All 14 counties had at least one bobcat with antibodies to T. gondii.     

Eradication of Feral Cats (Felis catus) from Gabo Island, south-east Victoria

Summary Concerns about the effects of predation by Feral Cats ( Felis catus ) on native fauna, particularly breeding seabirds, precipitated a decision in 1987 to control and eventually eradicate cats from Gabo Island. The size of the population prior to control was at least 30 animals. A control programme, undertaken between 1987 and 1991, centred on shooting, trapping and an extensive 1080 poison-baiting programme. Trapping and shooting were ineffectual. Poisoning was the most successful and effective technique for the rapid and widespread reduction in the Feral Cat population on Gabo Island. The effectiveness of dead 1-day-old chickens as a poison carrier was demonstrated. Effective poison baiting was attributed to bait selection and strategic timing of baiting to periods when prey was at low levels. Outcomes from the trapping programme and post-control monitoring strongly suggested that the cat population had been reduced to only two or three animals, possibly of the same sex. Monitoring between 1992 and 1998 failed to record any evidence of cats, indicating that the cats remaining after poison baiting had been unable to sustain a viable population. On the basis of the available evidence, Feral Cats appear to have been successfully eradicated from Gabo Island.     

Isolation and genetic characterization of Toxoplasma gondii from raccoons (Procyon lotor), cats (Felis domesticus), striped skunk (Mephitis mephitis), black bear (Ursus americanus), and cougar (Puma concolor) from Canada

Viable Toxoplasma gondii was isolated by bioassay in mice from tissues of 2 feral cats (Felis domesticus), 2 raccoons (Procyon lotor), a skunk (Mephitis mephitis) trapped in remote locations in Manitoba, Canada, and a black bear (Ursus americanus) from Kuujjuaq, northern Quebec, Canada. Genotyping of these T. gondii isolates using polymorphisms at 10 nuclear markers including SAGI, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico revealed 4 genotypes. None of the isolates was clonal archetypal Types I, II, and III found in the United States. These results are in contrast with the Type II genotype that is widespread in domestic animals and humans throughout the United States and Europe. This is the first genotyping of T. gondii isolates from this part of North America.     

Seroprevalence of Toxoplasma gondii from free-ranging black bears ( Ursus americanus ) from Florida

Toxoplasma gondii is a significant worldwide parasitic protozoan. In the present study, prevalence of antibodies of T. gondii was examined from 29 free-ranging black bears ( Ursus americanus ) from south-central Florida where the host species was listed as state threatened during this project. Overall T. gondii prevalence was found to be 44.8%, specifically 46.2% in male and 43.8% in female U. americanus , using a modified agglutination test (1:25 titer). Seroprevalence differences between sexes were not significant (P > 0.05). Results of the present study add supportive data to the growing body of evidence suggesting that U. americanus has one of the highest T. gondii seroprevalences among all known intermediate hosts. In addition, our data emphasize the importance of understanding parasitic disease dynamics from a conservation perspective.     

Transmission of Toxoplasma gondii in an urban population of domestic cats (Felis catus)

Toxoplasma gondii is a protozoan parasite that infects humans and animal species worldwide. The relative importance of each potential transmission route in the complex life cycle of this coccidia is largely unknown, due to the lack of studies taking into account all routes simultaneously. In this study, we analyzed the transmission of T. gondii in an urban population of stray cats captured between 1993 and 2004. Analyzing prevalence, our aim was to determine which factors influence transmission in this population. Specific anti-T. gondii IgG antibodies were detected using the modified agglutination test. Firstly, we analyzed the kinetics of antibody titers in cats captured several times, using mixed linear models and correspondence analysis. We showed that antibody titers did not vary significantly with time and that titer 40 was the best threshold to separate individuals into two serological groups. Overall, prevalence was only 18.6%, thus transmission of T. gondii is infrequent in this population. As expected, a highly significant association was detected between age and presence of IgG antibodies. Prevalence was lowest in kittens aged 3-4 months, suggesting that newborn kittens may carry maternal antibodies and that vertical transmission is rare. After taking into account the effect of age, logistic regression showed that antibody carriage was related to factors that possibly related to the survival of oocysts: localization in the study site, origin of the cats, maximal temperatures and rain. Our results suggest that in this population, vertical transmission is rare, low predation limits prevalence, and oocyst survival is a determining factor in the risk of infection. We discuss the more general importance of conditions determining oocyst survival in the life cycle of T. gondii.     

Seroprevalence of antibodies to Toxoplasma gondii in lynx (Lynx canadensis) and bobcats (Lynx rufus) from Québec, Canada.

The seroprevalence of antibodies to Toxoplasma gondii was investigated in trapped lynx (Lynx canadensis) and bobcats (Lynx rufus) from Québec, Canada. Forty-seven of 106 (44%) lynx and 4 of 10 (40%) bobcats had positive titers for T. gondii (> or = 25) by means of the modified agglutination test incorporating mercaptoethanol and formalin-fixed tachyzoites. Seroprevalence was significantly higher (P < 0.0001) in adult lynx than in juvenile lynx. The presence of antibodies to T. gondii in lynx and bobcats suggests that this organism is widespread in the wild and that exposure to wild felids and game animals from Québec may represent a potential source of infection for humans.     

Biologic and molecular characteristics of Toxoplasma gondii isolates from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), black-winged lory (Eos cyanogenia), and cats (Felis catus)

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered virulent to outbred mice, whereas Type II and III isolates are not. In the present report, viable T. gondii was isolated for the first time from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), and black-winged lory (Eos cyanogenia). For the isolation of T. gondii, tissues were bioassayed in mice, and genotyping was based on the SAG2 locus. Toxoplasma gondii was isolated from 3 of 6 skunks, 1 of 4 Canada geese, and 2 of 2 feral cats (Felis catus) from Mississippi. All donor animals were asymptomatic. Viable T. gondii was also isolated from 5 of 5 lories that had died of acute toxoplasmosis in an aviary in South Carolina. Genotypes of T. gondii isolates were Type III (all skunks, lories, and the goose) and Type II (both cats). All 5 Type III isolates from birds and 2 of the 3 isolates from skunks were mouse virulent.     

Prevalence and Genetic Characterization of Toxoplasma gondii in House Sparrows (Passer domesticus) in Lanzhou,China

The prevalence of Toxoplasma gondii infection in birds has epidemiological significance because birds are indeed considered as a good indicator of environmental contamination by T. gondii oocysts. In this study, the prevalence of T. gondii in 313 house sparrows in Lanzhou, northwestern China was assayed by the modified agglutination test (MAT). Antibodies to T. gondii were positive in 39 (12.46%) of 313 samples (MAT titer ≥ 1:5). Tissues of heart, brain, and lung from the 39 seropositive house sparrows were tested for T. gondii DNA, 11 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 9 genetic markers, including 8 nuclear loci, i.e., SAG1, 5''- and 3''-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8 and an apicoplast locus Apico. Of them, 4 isolates were genotyped with complete data for all loci, and 2 genotypes (Type II variants; ToxoDB #3 and a new genotype) were identified. These results showed that there is a potential risk for human infection with T. gondii in this region. To our knowledge, this is the first report of T. gondii seroprevalence in house sparrows in China.     

The Legal Status of Cats in New Zealand: A Perspective on the Welfare of Companion,Stray, and Feral Domestic Cats (Felis catus)

Pinpointing and safeguarding the welfare status of domestic cats is problematic, especially in New Zealand where cats are introduced predators with significant impact on indigenous fauna. Usually the identification of welfare status depends on conservational, legal, and public attitudes that are often contrasting. Cats may rapidly transgress definitions placed on them, confounding attempts to categorize them. In 1 generation, cats can move from a human-dependent state (“stray” or “companion”) to wild (“feral”). Often this categorization uses arbitrary behavioral and or situational parameters; consequent treatment and welfare protection for these cats are similarly affected. Terminology used to describe cats is not equitable across research. However, the New Zealand Animal Welfare (Companion Cats) Code of Welfare 2007 seeks to create a new definition of the terms companion, stray, and feral. It distinguishes between cats who live within and without human social constructs. This legislation mandates that cats in human environments or indirectly dependent on humans cannot be classified as feral. Such definitions may prove vital when safeguarding the welfare of free-living domestic cats and cat colonies.     

The Genetic Integrity of the Ex Situ Population of the European Wildcat (Felis silvestris silvestris) Is Seriously Threatened by Introgression from Domestic Cats (Felis silvestris catus)

Studies on the genetic diversity and relatedness of zoo populations are crucial for implementing successful breeding programmes. The European wildcat, Felis s. silvestris, is subject to intensive conservation measures, including captive breeding and reintroduction. We here present the first systematic genetic analysis of the captive population of Felis s. silvestris in comparison with a natural wild population. We used microsatellites and mtDNA sequencing to assess genetic diversity, structure and integrity of the ex situ population. Our results show that the ex situ population of the European wildcat is highly structured and that it has a higher genetic diversity than the studied wild population. Some genetic clusters matched the breeding lines of certain zoos or groups of zoos that often exchanged individuals. Two mitochondrial haplotype groups were detected in the in situ populations, one of which was closely related to the most common haplotype found in domestic cats, suggesting past introgression in the wild. Although native haplotypes were also found in the captive population, the majority (68%) of captive individuals shared a common mtDNA haplotype with the domestic cat (Felis s. catus). Only six captive individuals (7.7%) were assigned as wildcats in the STRUCTURE analysis (at K = 2), two of which had domestic cat mtDNA haplotypes and only two captive individuals were assigned as purebred wildcats by NewHybrids. These results suggest that the high genetic diversity of the captive population has been caused by admixture with domestic cats. Therefore, the captive population cannot be recommended for further breeding and reintroduction.     

Zoonotic Intestinal Trematodes in Stray Cats (Felis catus) from Riverside Areas of the Republic of Korea

The present study was performed to survey the infection status of zoonotic intestinal trematode (ZIT) in stray cats from 5 major riverside areas in the Republic of Korea. Total 400 stray cats were captured with live-traps in riverside areas of Seomjingang (‘gang’ means river) (203 cats) from June to October 2010, and of Yeongsangang (41), Nakdonggang (57), Geumgang (38), and Hangang (61 cats) from June to October 2011, respectively. Small intestines resected from cats were opened with a pair of scissors in a beaker with 0.85% saline and examined with naked eyes and under a stereomicroscope. More than 16 ZIT species were detected in 188 (92.6%) cats from Seomjingang areas, and the number of worms recovered was 111 per cat infected. In cats from riverside areas of Yeongsangang, Nakdonggang, Geumgang, and Hangang, more than 9, 8, 3, and 5 ZIT species were recovered, and the worm burdens were 13, 42, 11, and 56 specimens per infected cat, respectively. As the members of family Heterophyidae, more than 10 species, i.e., Metagonimus spp., Pygidiopsis summa, Heterophyes nocens, Stellantchasmus falcatus, Heterophyopsis continua, Acanthotrema felis, Centrocestus armatus, Procerovum varium, Cryptocotyle concava, and Stictodora lari, were recovered. More than 5 species of echinostomes, i.e., Echinostoma hortense, Echinochasmus japonicus, Echinochasmus sp., Echinoparyphium sp., and unidentified larval echinostomes, were collected. Plagiorchis spp. were detected in cats from areas of Seomjin-gang and Yeongsangang. From the above results, it has been confirmed that stray cats in 5 major riverside areas of Korea are highly infected with various species of ZITs.     

Eimeria albertensis n.sp. and E. borealis n.sp. (Sporozoa: Eimeriidae) in Black Bears Ursus americanus from Alberta

SYNOPSIS Eimeria albertensis n.sp. and E. borealis n.sp. are described from black bears Ursus americanus from Alberta, Canada. Oocysts of E. albertensis are ellipsoidal, 41.5 (36.5-43.8) by 21.7 (19.0-22.5) μ, double walled and have a distinct micropyle; sporocysts elongate-ovoid, 14.7 (10.9-18.7) by 7.7 (7.2-10.9) μ. Oocysts of E. borealis are ellipsoidal to slightly concave, 30.2 (28.8-32.8) by 14.8 (14.6-15.8) μ, and double walled; micropyle indistinct; sporocysts ovoid, 10.1 (7.3-11.5) by 6.1 (3.6-7.3) μ.     

Prevalence of antibodies against Toxoplasma gondii in polar bears (Ursus maritimus) from Svalbard and East Greenland

Serum samples from 419 polar bears (Ursus maritimus) from Svalbard and the Barents Sea (collected 1990-2000) and 108 polar bears from East Greenland (collected 1999-2004) were assayed for antibodies against Toxoplasma gondii using the modified agglutination test. Antibody prevalences were 3.6% among cubs dependent on their mothers and 21.4% among subadults and adults. Among subadults and adults there was an interaction between population and sex, with similar prevalences among females (Svalbard = 19.5%, Greenland = 18.0%), but a high frequency among Svalbard males (28.7%) as compared to Greenland males (5.8%). The pattern was also significant after correcting for differences in age distribution. The sex-population interaction term is believed to be connected to area- and sex-specific feeding ecology. The prevalences of antibodies against T. gondii in Svalbard and Greenland were high compared to previously reported findings in polar bears from Russian and Alaskan areas.     

Kinship and social structure of bobcats (Lynx rufus) inferred from microsatellite and radio-telemetry data

Kinship analysis using 12 microsatellites was compared with radio-telemetry data to examine the social structure of bobcats Lynx rufus in southern Texas. Genetically identified kinship relationships combined with capture data were used to reconstruct pedigrees. Three family groups were constructed from parent/offspring pairs identified from shared alleles. All parents identified by genetic analysis had established home ranges. Individuals with no distinct home ranges were not genetically observed to have offspring among the bobcats sampled. This suggests that establishing a home range is necessary for bobcats to breed. Of three identified male offspring and three identified female offspring, two female offspring were philopatric. These females became a part of the breeding population in their natal area. Among sibling pairs that included nine female and four male individuals, four females and one male were residents suggesting male-biased dispersal.     

Isolation and genetic characterization of Toxoplasma gondii from striped dolphin (Stenella coeruleoalba) from Costa Rica

Toxoplasma gondii infection in marine mammals is of interest because of mortality and mode of transmission. It has been suggested that marine mammals become infected with T. gondii oocysts washed from land to the sea. We report the isolation and genetic characterization of viable T. gondii from a striped dolphin (Stenella coeruleoalba), the first time from this host. An adult female dolphin was found stranded on the Pacific Coast of Costa Rica, and the animal died the next day. The dolphin had a high (1:6400) antibody titer to T. gondii in the modified agglutination test. Severe nonsuppurative meningoencephalomyelitis was found in its brain and spinal cord, but T. gondii was not found in histological sections of the dolphin. Portions of its brain and the heart were bioassayed in mice for the isolation of T. gondii. Viable T. gondii was isolated from the brain, but not from the heart, of the dolphin. A cat fed mice infected with the dolphin isolate (designated TgSdCol) shed oocysts. Genomic DNA from tachyzoites of this isolate was used for genotyping at 10 genetic loci, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and this TgSdCo1 isolate was found to be Type II.     

Molecular Cloning,Recombinant Expression,and Funtional Characterization of APRIL (TNFSF13) in Cat (Felis catus)

A proliferation-inducing ligand (APRIL) is a critical member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the cDNA of cat APRIL (cAPRIL) was successfully amplified. Sequence analysis showed that the open reading frame (ORF) of cAPRIL contains a putative furin protease cleavage site (R-R-K-R), a conserved putative N-glycosylation site (Asn¹²⁴), and two conservative cysteine residues (Cys¹⁹⁶ and Cys²¹¹). Real-time quantitative PCR (qPCR) analysis revealed that cAPRIL could be detected in various tissues. The phylogenetic analysis and predicted three dimensional (3D) structure revealed that it is similar to its counterparts. The extracellular soluble domain of the cAPRIL (csAPRIL) fragment was cloned into the expression vector pET43.1a. SDS-PAGE and Western blotting analysis indicated a high-level expression of csAPRIL protein in Escherichia coli BL21 (DE3). MTT assays revealed that purified recombinant csAPRIL protein was able to stimulate proliferation of mouse B-cells. These findings indicate that cAPRIL plays an important role in proliferation of B-cells and provide the basis for investigation on the roles of APRIL in this important domestic species.     

Characterization of Toxoplasma gondii from raccoons (Procyon lotor), coyotes (Canis latrans), and striped skunks (Mephitis mephitis) in Wisconsin identified several atypical genotypes

During 2005-2006, sera and tissues from raccoons (Procyon lotor), coyotes (Canis latrans), and skunks (Mephitis mephitis) from the state of Wisconsin were tested for Toxoplasma gondii infection. Antibodies to T. gondii were found in 32 of 54 (59.2%) raccoons, 18 of 35 (51.4%) coyotes, and 5 of 7 (71.4%) skunks using the modified agglutination test and a cut-off titer of 1:20. Pooled tissues (brains, hearts, and tongues) from 30 raccoons, 15 coyotes, and 1 skunk were bioassayed for T. gondii infection in mice or cats. Viable T. gondii was isolated from 5 of 30 (16.7%) raccoons, 6 of 15 (40.0%) coyotes, and the skunk. Genetic characterization of the 12 parasite isolates by multilocus PCR-RFLP markers revealed 6 different genotypes including 5 atypical and I archetypal II lineages. The results indicate the prevalence of T. gondii in wildlife mammals is high and that these animals may serve as an important reservoir for transmission of T. gondii.