Camila K. Cerqueira-Cézar

Toxoplasmosis in geese and detection of two new atypical Toxoplasma gondii strains from naturally infected Canada geese (Branta canadensis)

Wild birds are important in the epidemiology of toxoplasmosis because they can serve as reservoir hosts, and vectors of zoonotic pathogens including Toxoplasma gondii. Canada goose (Branta canadensis) is the most widespread geese in North America. Little is known concerning T. gondii infection in both migratory, and local resident populations of Canada geese. Here, we evaluated the seroprevalence, isolation, and genetic characterization of viable T. gondii isolates from a migratory population of Canada geese. Antibodies against T. gondii were detected in 12 of 169 Canada geese using the modified agglutination test (MAT, cutoff 1:25). The hearts of 12 seropositive geese were bioassayed in mice for isolation of T. gondii. Viable parasites were isolated from eight. One isolate was obtained from a seropositive goose by both bioassays in mice, and in a cat; the cat fed infected heart excreted T. gondii oocysts. Additionally, one isolate was obtained from a pool of four seronegative (<1:25) geese by bioassay in a cat. The T. gondii isolates were further propagated in cell culture, and DNA extracted from cell culture-derived tachyzoites were characterized using 10 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genetic markers (SAG1, 5′ and 3′SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed five different genotypes. ToxoDB PCR-RFLP genotype #1 (type II) in one isolate, genotype #2 (type III) in four isolates, genotype #4 in two isolates, and two new genotypes (ToxoDB PCR-RFLP genotype #266 in one isolate and #267 in one isolate) were identified. These results indicate genetic diversity of T. gondii strains in the Canada geese, and this migratory bird might provide a mechanism of T. gondii transmission at great distances from where an infection was acquired.

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.

Environmental and behavioral changes may influence the exposure of an Arctic apex predator to pathogens and contaminants

Recent decline of sea ice habitat has coincided with increased use of land by polar bears (Ursus maritimus) from the southern Beaufort Sea (SB), which may alter the risks of exposure to pathogens and contaminants. We assayed blood samples from SB polar bears to assess prior exposure to the pathogens Brucella spp., Toxoplasma gondii, Coxiella burnetii, Francisella tularensis, and Neospora caninum, estimate concentrations of persistent organic pollutants (POPs), and evaluate risk factors associated with exposure to pathogens and POPs. We found that seroprevalence of Brucella spp. and T. gondii antibodies likely increased through time, and provide the first evidence of exposure of polar bears to C. burnetii, N. caninum, and F. tularensis. Additionally, the odds of exposure to T. gondii were greater for bears that used land than for bears that remained on the sea ice during summer and fall, while mean concentrations of the POP chlordane (ΣCHL) were lower for land-based bears. Changes in polar bear behavior brought about by climate-induced modifications to the Arctic marine ecosystem may increase exposure risk to certain pathogens and alter contaminant exposure pathways.

Seroprevalence of Neospora caninum in feral swine (Sus scrofa) in the United States

The protozoon Neospora caninum is a major cause of abortion in cattle worldwide. Canids (Canis familiaris, Canis latrans, Canis lupus) are definitive hosts whereas many other animal species, including pigs, are intermediate hosts for the parasite. Between 2012 and 2014, serum samples from 1059 feral swine (Sus scrofa) from 29 states of the USA were tested for N. caninum antibodies, using the N. caninum agglutination test (NAT). Of these, 159 (15.0%) feral pigs from 21 states tested positive, with a range of titers of 1:25 (cut-off) (n=153), 1:200 (1), 1:400 (1), 1:800 (3) and 1:3200 (1). Results indicate widespread exposure of feral swine to N. caninum infection across the USA.

Seroepidemiologic study on the prevalence of Toxoplasma gondii and Trichinella spp. infections in black bears (Ursus americanus) in Pennsylvania, USA.

The protozoan Toxoplasma gondii and the metazoan Trichinella spp. infect virtually all warm-blooded animals, including birds, humans, livestock, and marine mammals. Both parasitic infections can cause serious illness in human beings and can be acquired by ingesting under-cooked meat harboring infective stages. Approximately 3500 black bears (Ursus americanus) are legally-harvested each year in Pennsylvania, USA during the November hunting season. Among animals found infected with T. gondii, the prevalence of T. gondii is the highest among black bears in the USA; however, little is currently known of epidemiology of toxoplasmosis in this host species. Serum samples were collected during the winters of 2015 and 2016 from adult female bears and their nursing cubs or yearlings while they were still in their dens. Additionally, archived sera from bear samples collected throughout the year, including hunter-harvested bears in November and trapped bears in the summer, were serologically tested. Antibodies to T. gondii were assayed by the modified agglutination test (MAT, cut-off 1:25) and antibodies to Trichinella spp. were assayed using a commercial enzyme-linked immunosorbent assay (ELISA). Overall, T. gondii antibodies were found in 87.6% (206/235) of adults, and 44.1% (30/68) of yearlings. In March 2015/2016 sampling, antibodies to T. gondii were found in 94% (30/32) adult female bears while in their den. Antibodies were detected in 5% (3/66) of the nursing cubs in the dens of these sows. One positive cub had a MAT titer of 1:160 and two were positive at the 1:25 dilution but not at 1:50. The adult females of these cubs had MAT titers ranging from 1:400 to 1:3200. Antibodies to Trichinella spp. were found in 3% (6/181) of adults and 3.6% (1/28) of yearlings; these 7 bears were also seropositive for T. gondii. No antibodies to Trichinella spp. were detected in the sera of 44 nursing cubs tested. The finding of T. gondii antibodies in only 3 of 66 cubs, and higher antibody titers in their respective sows indicates that the colostrally-acquired antibodies wane to undetectable levels by 8-10 weeks, while the cubs are still in the den. The results indicate that there is no transplacental transmission of T. gondii, that antibodies acquired from colostrum are largely undetectable by the time cubs emerge from the den, and nearly that 50% of bears acquire infection postnatally by 10 months of age. This is the first report of disappearance of transcolostral antibodies of any infection in bears.

Dissection of the in vitro developmental program of Hammondia hammondi reveals a link between stress sensitivity and life cycle flexibility in Toxoplasma gondii

Most eukaryotic parasites are obligately heteroxenous, requiring sequential infection of different host species in order to survive. Toxoplasma gondii is a rare exception to this rule, having a uniquely facultative heteroxenous life cycle. To understand the origins of this phenomenon, we compared development and stress responses in T. gondii to those of its its obligately heteroxenous relative, Hammondia hammondi and have identified multiple H. hammondi growth states that are distinct from those in T. gondii. Of these, the most dramatic difference was that H. hammondi was refractory to stressors that robustly induce cyst formation in T. gondii, and this was reflected most dramatically in its unchanging transcriptome after stress exposure. We also found that H. hammondi could be propagated in vitro for up to 8 days post-excystation, and we exploited this to generate the first ever transgenic H. hammondi line. Overall our data show that H. hammondi zoites grow as stringently regulated, unique life stages that are distinct from T. gondii tachyzoites, and implicate stress sensitivity as a potential developmental innovation that increased the flexibility of the T. gondii life cycle.

An update on Toxoplasma gondii infections in northern sea otters (Enhydra lutris kenyoni) from Washington State, USA

Toxoplasmosis in marine mammals is epidemiologically and clinically important. Toxoplasma gondii antibodies (by modified agglutination test, cut-off ≥1:25) were detected in serum of 65 of 70 (92.9%) northern sea otters (Enhydra lutris kenyoni) from Washington State, USA. Brains and/or muscles of 44 sea otters were bioassayed in mice (INF-γ knock-out [KO], Swiss Webster outbred [SW]) and viable T. gondii was isolated from 22 of 44 (50%); T. gondii strains were lethal to KO mice but not SW mice. These T. gondii isolates were further propagated in cell culture. Multi-locus PCR-RFLP genotyping of cell culture-derived tachyzoites revealed four different genotypes among 22 isolates including ToxoDB PCR-RFLP genotype #5 (14 isolates), #1 (three isolates), #3 (four isolates), and #167 (one isolate). PCR-DNA sequencing based genotyping using polymorphic gene GRA6 revealed one of four different alleles. Among the 14 RFLP genotype #5 strains, 10 have GRA6 sequences that match with the Type A, one match with the Type X, two strains did not generate sequence data, and one strain had double peaks at known polymorphic sites indicating a mixed infection. The seven strains belong to genotypes #1 and #3, all have identical sequences to T. gondii Type II reference isolate ME49. Genotype #167 strain has identical sequence to Type I reference strain. In summary, we observed high seroprevalence, and high rate of isolation of T. gondii from northern sea otters and predominant genotype #5 that has been previously reported a dominant and widespread strain among terrestrial wildlife in North America. GRA6 sequence analysis of the genotype #5 isolates indicated the dominance of Type A lineage in sea otters in Washington State.

Role of Rats (Rattus norvegicus) in the Epidemiology of Toxoplasma gondii Infection in Grenada, West Indies

Abstract Rodents are known to be reservoir hosts of Toxoplasma gondii infection for other animals, such as cats and pigs. From February to July 2017, 167 rats (Rattus norvegicus) were trapped in Grenada, and serum, heart, skeletal muscle, and brain were examined for T. gondii infection by serological examination (modified agglutination test, 1:25) for T. gondii antibodies and for viable parasites by bioassay in mice. Samples of heart, skeletal muscle, and brain of all rats were bioassayed in Swiss Webster (SW) outbred albino mice and interferon gamma gene knockout (KO) mice. Toxoplasma gondii was isolated from heart and brain from 1 rat; this was the only seropositive rat. The T. gondii strain was avirulent for SW mice but killed KO mice. Tissue cysts were detected in the brains of SW mice, and tachyzoites were detected in the lungs of KO mice that died of acute toxoplasmosis. The strain was propagated in cell culture, and DNA derived from cell-cultured tachyzoites was genotyped using the 10 PCR restriction fragment length polymorphisms (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The strain was a clonal Type III (ToxoDB genotype no. 2) strain. Although the prevalence of T. gondii in humans and animals in Grenada is high, rats seem to have little importance in the transmission of T. gondii on this island.

Acute fatal sarcocystosis hepatitis in an Indo-Pacific bottlenose dolphin (Tursiops aduncus) in Hong Kong

Unlike most species in the genus Sarcocystis, Sarcocystis canis has a broad intermediate host range. Its life cycle is incompletely known and most reports are from the USA. Here we report fatal hepatitis in a 4year old male Indo-Pacific bottlenose dolphin (Tursiops aduncus) from Hong Kong associated with a S. canis-like infection. Diagnosis was made based on clinical presentation, histopathology, transmission electron microscopy (TEM), and molecular characterization. Microscopically, S. canis-like like infection was confined to the liver. Immature and mature schizonts were found in hepatocytes and the parasite was associated with generalized hepatic necrosis. By TEM, schizonts divided by endopolygeny, and merozoites lacked rhoptries. Molecular characterization of parasites present in liver and brain tissues at the cox1 gene showed a high degree of identity (97-98%) and clustered together with Sarcocystis canis, S. lutrae, S. arctica, S. speeri, S. turdusi, and S. rileyi in a phylogenetic study. This is the first report of S. canis-like infection from Asia.

Bobcats (Lynx rufus) are natural definitive host of Besnoitia darlingi

Bovine besnoitiosis, caused by Besnoitia besnoiti, is an economically important disease of cattle in many countries but its transmission remains a mystery. Wild felids are suspected to be its definitive hosts. The domestic cat (Felis catus) is known experimental definitive host for Besnoitia species of rodents. Here, we report for Besnoitia darlingi the first identification of a natural definitive host, the bobcat (Lynx rufus). Oocysts resembling Toxoplasma gondii (unsporulated; 10.9±0.8×12.1±0.2μm; n=5) were detected microscopically in the feces of two of 25 free ranging wild bobcats from Mississippi, USA. After detailed investigation, we identified these oocysts as B. darlingi and not T. gondii. The IFN-γ gene knockout (KO) mice fed oocysts from bobcats died of acute besnoitiosis and tachyzoites were found in their tissues. Oocysts were also mildly pathogenic to outbred Swiss Webster mice (SW) (Mus musculus). The SW mice fed oocysts became ill but generally survived and developed characteristic thick-walled Besnoitia tissue cysts in their tongue and heart muscles and brains. Two laboratory-raised domestic cats (Felis catus) excreted B. darlingi oocysts after ingesting murine tissues infected with bobcat-derived oocysts. The parasite was successfully cultivated in African green monkey kidney fibroblast cells (CV-1 cell line) seeded with infected murine tissue homogenate. The multilocus PCR-DNA sequencing (18S rDNA, 28S rDNA, and ITS-1) from culture-derived tachyzoites confirmed the parasite as B. darlingi. Our results suggest that bobcats may be an important link in the sylvatic cycle of Besnoitia species and bioassay or molecular tests are needed to differentiate Toxoplasma gondii-like oocysts in feces of felids, both domestic and wild cats.