Shanti Choudhary - Academia.edu (original) (raw)

Papers by Shanti Choudhary

Journal of Pure and Applied Microbiology, Nov 29, 2023

JDS Communications

• A method for culturing hepatic cells from abattoir-derived dairy cow liver is presented. • This... more • A method for culturing hepatic cells from abattoir-derived dairy cow liver is presented. • This method utilizes adult dairy cow tissue that is readily available. • Because the culture comprises all hepatic cell types in comparable proportions to those in the live dairy cow, we expect this method to be more representative of in vivo fatty liver disease. • Our method is simple, novel, consistent with other cell models, and consistent with fatty liver disease in dairy cows during the periparturient period. • We present a novel method to pursue mechanistic studies of fatty liver in the periparturient dairy cow.

Veterinary Parasitology, Sep 1, 2013

Neosporosis is an important cause of bovine abortion worldwide. Many aspects of transmission of N... more Neosporosis is an important cause of bovine abortion worldwide. Many aspects of transmission of Neospora caninum in nature are unknown. The white-tailed deer (Odocoileus virginianus) is considered one of the most important wildlife reservoirs of N. caninum in the USA. During the hunting seasons of 2008, 2009, and 2010, brains of 155 white-tailed deer fetuses were bioassayed in mice for protozoal isolation. Viable N. caninum (NcWT-DMn1, NcWTDMn2) was isolated from the brains of two fetuses by bioassays in mice, and subsequent propagation in cell culture. Dams of these two infected fetuses had antibodies to N. caninum by Neospora agglutination test at 1:100 serum dilution. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates with Nc5 PCR confirmed diagnosis. Results prove congenital transmission of N. caninum in the white tailed deer for the first time.

Veterinary Parasitology, Sep 1, 2013

Black bears (Ursus americanus) are hosts for two important zoonotic parasites, Toxoplasma gondii ... more Black bears (Ursus americanus) are hosts for two important zoonotic parasites, Toxoplasma gondii and Trichinella spp. and bears are hunted for human consumption in the USA. Little is known of the genetic diversity of T. gondii circulating in wildlife. In the present study, antibodies to T. gondii were found in juice from tongues of 17 (25.7%) of 66 wild black bear from Maryland during the hunting season of 2010 and 2011. Antibodies to T. gondii were assessed by the modified agglutination test. Tongues of 17 seropositive bears were bioassayed in mice and viable T. gondii was isolated from three samples. These three T. gondii isolates (TgBbMd1-3) were further propagated in cell culture and DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers (SAG1, 5-and 3-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Results revealed three genotypes. TgBbMd1 is a Type 12 strain (ToxoDB PCR-RFLP genotype #4) and TgBbMd2 is ToxoDB PCR-RFLP genotype #216, and TgBbMd3 is a Type II clonal strain (ToxoDB PCR-RFLP genotype #1). The isolate TgBbMd2 was highly virulent for outbred Swiss Webster mice; all infected mice died of acute toxoplasmosis. Results indicate that mouse virulent strains of T. gondii are circulating in wildlife in the USA. These 66 tongues in addition to tongues collected during hunts in previous years were further investigated for the presence of muscle lar

Veterinary Parasitology, Mar 1, 2014

Neospora caninum is a common cause of abortion in cattle worldwide. Canids, including the dog and... more Neospora caninum is a common cause of abortion in cattle worldwide. Canids, including the dog and the dingo (Canis familiaris), the coyote (Canis latrans), and the gray wolf (Canis lupus) are its definitive hosts that can excrete environmentally resistant oocysts in the environment, but also can act as intermediate hosts, harboring tissue stages of the parasite. In an attempt to isolate viable N. caninum from tissues of naturally infected wolves, brain and heart tissue from 109 wolves from Minnesota were bioassayed in mice. Viable N. caninum (NcWolfMn1, NcWolfMn2) was isolated from the brains of two wolves by bioassays in interferon gamma gene knockout mice. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates were analyzed by N. caninum-specific Nc5 polymerase chain reaction and confirmed diagnosis. This is the first report of isolation of N. caninum from tissues of any wild canid host.

Veterinary Parasitology, Nov 1, 2013

The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondi... more The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondii in freerange chickens from Grenada, West Indies. Using the modified agglutination test, antibodies to T. gondii were found in 39 (26.9%) of 145 free-range chickens with titers of 25 in 7 chickens, 50 in 6 chickens, 100 in 2 chickens, and 200 or higher in 24 chickens. The hearts of the 39 seropositive chickens were bioassayed in mice; viable T. gondii was isolated from 20 and further propagated in cell culture. Genotyping of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed 4 genotypes, including ToxoDB PCR-RFLP no. 2 (Type III), no. 7, no. 13, and no. 259 (new). These results indicated that T. gondii population genetics in free-range chickens seems to be moderately diverse with ToxoDB no. 2 (Type III) as the most frequent (15/20 ¼ 75%) compared to other genotypes in Grenada.

Journal of Parasitology, Oct 1, 2013

The ingestion of uncooked infected white-tailed deer (WTD) tissues can transmit Toxoplasma gondii... more The ingestion of uncooked infected white-tailed deer (WTD) tissues can transmit Toxoplasma gondii infection to humans and mesocarnivores, including cats. In the present study, we tested 264 WTD from New Jersey for T. gondii infection during the 2011-2012 hunting season. Serum samples were tested for antibodies to T. gondii by the modified agglutination test (cutoff titer, 25); 76 (28.7%) of 264 WTD were seropositive. Heart muscle samples from 64 seropositive WTD were digested in pepsin, and the digests were bioassayed for the isolation of T. gondii . Viable T. gondii was isolated in mice from the myocardium of 9 WTD; tachyzoites from infected mouse tissues were further propagated in cell culture. One of the 9 strains was highly virulent for outbred Swiss Webster mice. The DNA isolated from culture-derived tachyzoites of these 9 T. gondii isolates was characterized using 11 PCR-RFLP markers (SAG1, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Six genotypes were found, including ToxoDB genotype no. 2 (Type III), no. 3 (Type II variant), no. 4 (Type 12), no. 216, no. 220, and no. 221. The last 2 were new genotypes that were reported for the first time. This is the first report of T. gondii infection in deer from this region of the United States.

Veterinary Parasitology, Nov 1, 2013

Nothing is known of the genetic diversity of Toxoplasma gondii circulating in wildlife in Mexico.... more Nothing is known of the genetic diversity of Toxoplasma gondii circulating in wildlife in Mexico. In the present study, a mouse virulent T. gondii strain was isolated from the heart of a wild puma (Felis concolor). The puma was found roaming in outskirt of Durango City, Mexico and tranquilized for moving to a zoo. The puma died during translocation and a necropsy examination was performed. The puma had an antibody titer for T. gondii of 200 by the modified agglutination test. Its heart and brain tissue were bioassayed into 2 outbred Swiss Webster (SW) and 1 gamma interferon gene knockout (KO) mouse. The KO mouse and the 2 SW mice that became infected after inoculation with homogenate of puma heart died of acute toxoplasmosis 12, 19 and 20 days p.i. respectively and tachyzoites were found in lungs of all 3 mice. None of the 4 SW and 1 KO mouse inoculated with digest of the puma brain became infected with T. gondii. Tachyzoites from the lungs of mice were propagated in cell cultures. Tachyzoites from cell culture were inoculated into 5 SW; the mice died or had to be killed 14 days p.i. and a cat fed tissues of these mice shed T. gondii oocysts. Results of mortality and infectivity of tachyzoites and oocysts in SW mice indicated that the puma T. gondii strain (designated TgPumaMe1) was virulent for outbred mice. DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers (SAG1, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed a new genotype (ToxoDB PCR-RFLP #222). Isolation of atypical genotype T. gondii from wild puma indicates that mouse virulent strains are circulating in wildlife in Mexico.

Veterinary Parasitology, 2014

Donkeys (Equus asinus) are used as both companion and working animals throughout the world and in... more Donkeys (Equus asinus) are used as both companion and working animals throughout the world and in some countries, their meat and milk are used for human consumption. Here we report the first serological survey of Toxoplasma gondii in donkeys in the United States. Serum samples from 373 donkeys from eight farms in five states were tested for T. gondii antibodies by the modified agglutination test (MAT). Twenty-four of 373 (6.4%) of donkeys were seropositive, with MAT titers ranging from 25 to ≥ 200. All seropositive donkeys were Miniature breed. Seropositivity prevalence was 7.0% in female donkeys (20/282) and 4.1% in male donkeys (4/91). No donkeys less than 24 months of age (129) were seropositive, suggesting postnatal transmission of infection. Domestic cats were present on six of the eight farms. Three cats from one farm had MAT titers of 200. Viable T. gondii was isolated from the hearts of two cats, but not from brain tissues. Genotyping of isolate DNA extracted from culture-derived tachyzoites using 10 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico loci) revealed that both isolates were clonal Type II (ToxoDB PCR-RFLP genotype #1). This is the first serological survey for T. gondii in donkeys in the United States, and suggests that donkey milk and meat should be considered as a potential source for human infection. The role of barn cats in the transmission of T. gondii to donkeys on farms warrents further investigation.

Veterinary Parasitology, Feb 1, 2014

Recent studies indicated the predominance of Toxoplasma gondii haplogroup 12 in wildlife in USA. ... more Recent studies indicated the predominance of Toxoplasma gondii haplogroup 12 in wildlife in USA. But still little is known of the genetic diversity of this parasite circulating in wildlife. In the present study, we tested coyotes (Canis latrans), red foxes (Vulpes vulpes), white-tailed deer (Odocoileus virginianus), and geese (Branta canadensis) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 160 of 367 animals, including 92 (34.5%) of 266 coyotes, 49 (62.0%) of 79 white-tailed deer, 17 (85.0%) of 20 red fox, and two of two Canada geese tested by the modified agglutination test (cut off titer 1:25). Tissues from 105 seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 29 animals, including 10 of 53 coyotes, 11 of 16 foxes, 7 of 49 deer, and one of one goose. DNA isolated from culture-derived tachyzoites of these isolates was characterized initially using multilocus PCR-RFLP markers. Nine genotypes were revealed, including ToxoDB PCR-RFLP #1 (4 isolates), #2 (2 isolates), #3 (4 isolates), #4 (6 isolates), #5 (4 isolates), #54 (1 isolate), #141 (1 isolate), #143 (1 isolate), and #216 (6 isolates), indicating high genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of six T. gondii isolates (5 of #216 and #141) was determined in outbred Swiss Webster mice. Three of #216 and the #141 isolates were acute virulent to mice, and the other 2 #216 isolates were intermediate virulent. To determine the extent of genetic variation of these as well as a few recently reported virulent isolates from wildlife in North America, intron sequences were generated. Analysis of intron sequences and PCR-RFLP genotyping results *

Veterinary Parasitology, Sep 1, 2013

Recent studies indicate greater genetic variability among isolates of Toxoplasma gondii worldwide... more Recent studies indicate greater genetic variability among isolates of Toxoplasma gondii worldwide than previously thought. However, there is no information on genetic diversity of T. gondii from any host in Ethiopia. In the present study, genotyping was performed on viable T. gondii isolates by bioassays in mice from tissues and feces of 27 cats from Ethiopia. Viable T. gondii was isolated from hearts of 26 cats, feces alone of 1 cat, and feces and tissues of 6 cats; in total there were 33 isolates. Genotyping was performed on DNA from cell-cultured derived T. gondii tachyzoites and by using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Four genotypes were recognized, including ToxoDB #1 (Type II clonal, nine isolates), ToxoDB #2 (Type III, five isolates), Toxo DB #3 (Type II variant, ten isolates), and ToxoDB #20 (nine isolates). Of interest is the isolation of different genotypes from tissues and feces of two cats, suggesting re-infection or mixed strain T. gondii infection. These findings are of epidemiological significance with respect to shedding of oocysts by cats. This is the first report of genotyping of T. gondii from any host in Ethiopia.

Journal of Parasitology, Aug 1, 2013

Journal of Parasitology, Feb 1, 2013

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that ... more Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that excrete environmentally resistant oocysts in feces. In the present study, hearts, serum, and feces from 36 feral cats from Addis Ababa area, Ethiopia, were examined for T. gondii infection. Antibodies to T. gondii were determined with the modified agglutination test (MAT, cutoff 1:25); 33 cats were seropositive. Hearts of all 36 cats were homogenized, digested in pepsin, and bioassayed in mice. Feces were examined for T. gondii oocysts by bioassay in mice. Viable T. gondii was isolated from heart of 26 by bioassay in mice and from 25 seropositive and 1 seronegative cats. Toxoplasma gondii was isolated from feces (oocysts) by bioassay in mice. In total, viable T. gondii was isolated from 27 of the 36 cats, and these isolates were designated TgCatEt1 to TgCatEt27. The high prevalence of T. gondii oocysts in feces of 8 (19.4%) of 36 cats is of high epidemiologic significance. This is the first report of isolation of viable T. gondii from any host in Ethiopia.

Veterinary Parasitology, Nov 1, 2013

The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondi... more The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondii in freerange chickens from Grenada, West Indies. Using the modified agglutination test, antibodies to T. gondii were found in 39 (26.9%) of 145 free-range chickens with titers of 25 in 7 chickens, 50 in 6 chickens, 100 in 2 chickens, and 200 or higher in 24 chickens. The hearts of the 39 seropositive chickens were bioassayed in mice; viable T. gondii was isolated from 20 and further propagated in cell culture. Genotyping of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed 4 genotypes, including ToxoDB PCR-RFLP no. 2 (Type III), no. 7, no. 13, and no. 259 (new). These results indicated that T. gondii population genetics in free-range chickens seems to be moderately diverse with ToxoDB no. 2 (Type III) as the most frequent (15/20 ¼ 75%) compared to other genotypes in Grenada.

Veterinary Parasitology, 2013

Stray dogs are considered as sentinels in the epidemiology of Toxoplasma gondii because they are ... more Stray dogs are considered as sentinels in the epidemiology of Toxoplasma gondii because they are carnivores and eat variety of foods, including garbage. In the present study, tissues and sera of 51 stray dogs (Canis familiaris) from Giza, Egypt were examined for T. gondii infection. Sera were examined for antibodies to T. gondii by the modified agglutination test (MAT); 50 of 51 (98%) were seropositive with titers of 20 in four, 40 in four, 80 in one, 100 in eight, 200 in 17, 400 in 11, 800 or higher in five. Hearts of 43 seropositive dogs were bioassayed in mice. Viable T. gondii was isolated from 22 dogs; these isolates were designated TgDogEg1 to TgDogEg22. DNA isolated from cell culture derived tachyzoites of 22 isolates was genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed three genotypes and one mixed infection. The three genotypes are ToxoDB PCR-RFLP #2 (type III, four isolates), #3 (type II variant, 11 isolates), #20 (six isolates), 1 mixed infection. These results revealed the dominance of clonal type II, III and ToxoDB #20 lineages of T. gondii in stray dogs from Giza, Egypt.

Veterinary Parasitology, 2011

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. N... more The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum.

Journal of Parasitology, 2007

Two independently derived, exotoxin A-deficient (Tox-phenotype), nitrosoguanidine-induced mutants... more Two independently derived, exotoxin A-deficient (Tox-phenotype), nitrosoguanidine-induced mutants of Pseudomonas aeruginosa PAO 1 were isolated by using sensitive immunological assays. One mutant, designated PAOT1O, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT1O and the parental strain. The mutation in PAOT1O (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.

International Journal for Parasitology, 2011

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the pre... more Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5 0-and 3 0-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types.

Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 2012

Journal of Zoo and Wildlife Medicine, Dec 31, 2013

Little is known of the genetic diversity and epidemiology of Toxoplasma gondii infection in wildl... more Little is known of the genetic diversity and epidemiology of Toxoplasma gondii infection in wildlife in Caribbean Islands. The prevalence and genetic diversity of T. gondii in mongooses (Herpestes auropunctatus) was investigated. During 2011 and 2012, 91 mongooses were trapped in different parts of Grenada, bled, euthanized, and examined at necropsy. Antibodies to T. gondii were found in 27 mongooses tested by the modified agglutination test (cut-off titer 25). Muscles (heart, tongue, neck) of 25 of the seropositive mongooses were bioassayed for T. gondii infection in mice. Viable T. gondii was isolated by bioassay in mice from four mongooses with MAT titers of 1:50 in two, 1:200 for one, and 1:400 for one mongoose. The four T. gondii isolates were further propagated in cell culture. Strain typing of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed one isolate belongs to the Type III (ToxoDB #2) lineage, two to ToxoDB#7 lineage, and one to the ToxoDB #216 lineage. This is the first report of T. gondii isolation and genotyping in H. auropunctatus worldwide.

Journal of Pure and Applied Microbiology, Nov 29, 2023

JDS Communications

• A method for culturing hepatic cells from abattoir-derived dairy cow liver is presented. • This... more • A method for culturing hepatic cells from abattoir-derived dairy cow liver is presented. • This method utilizes adult dairy cow tissue that is readily available. • Because the culture comprises all hepatic cell types in comparable proportions to those in the live dairy cow, we expect this method to be more representative of in vivo fatty liver disease. • Our method is simple, novel, consistent with other cell models, and consistent with fatty liver disease in dairy cows during the periparturient period. • We present a novel method to pursue mechanistic studies of fatty liver in the periparturient dairy cow.

Veterinary Parasitology, Sep 1, 2013

Neosporosis is an important cause of bovine abortion worldwide. Many aspects of transmission of N... more Neosporosis is an important cause of bovine abortion worldwide. Many aspects of transmission of Neospora caninum in nature are unknown. The white-tailed deer (Odocoileus virginianus) is considered one of the most important wildlife reservoirs of N. caninum in the USA. During the hunting seasons of 2008, 2009, and 2010, brains of 155 white-tailed deer fetuses were bioassayed in mice for protozoal isolation. Viable N. caninum (NcWT-DMn1, NcWTDMn2) was isolated from the brains of two fetuses by bioassays in mice, and subsequent propagation in cell culture. Dams of these two infected fetuses had antibodies to N. caninum by Neospora agglutination test at 1:100 serum dilution. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates with Nc5 PCR confirmed diagnosis. Results prove congenital transmission of N. caninum in the white tailed deer for the first time.

Veterinary Parasitology, Sep 1, 2013

Black bears (Ursus americanus) are hosts for two important zoonotic parasites, Toxoplasma gondii ... more Black bears (Ursus americanus) are hosts for two important zoonotic parasites, Toxoplasma gondii and Trichinella spp. and bears are hunted for human consumption in the USA. Little is known of the genetic diversity of T. gondii circulating in wildlife. In the present study, antibodies to T. gondii were found in juice from tongues of 17 (25.7%) of 66 wild black bear from Maryland during the hunting season of 2010 and 2011. Antibodies to T. gondii were assessed by the modified agglutination test. Tongues of 17 seropositive bears were bioassayed in mice and viable T. gondii was isolated from three samples. These three T. gondii isolates (TgBbMd1-3) were further propagated in cell culture and DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers (SAG1, 5-and 3-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Results revealed three genotypes. TgBbMd1 is a Type 12 strain (ToxoDB PCR-RFLP genotype #4) and TgBbMd2 is ToxoDB PCR-RFLP genotype #216, and TgBbMd3 is a Type II clonal strain (ToxoDB PCR-RFLP genotype #1). The isolate TgBbMd2 was highly virulent for outbred Swiss Webster mice; all infected mice died of acute toxoplasmosis. Results indicate that mouse virulent strains of T. gondii are circulating in wildlife in the USA. These 66 tongues in addition to tongues collected during hunts in previous years were further investigated for the presence of muscle lar

Veterinary Parasitology, Mar 1, 2014

Neospora caninum is a common cause of abortion in cattle worldwide. Canids, including the dog and... more Neospora caninum is a common cause of abortion in cattle worldwide. Canids, including the dog and the dingo (Canis familiaris), the coyote (Canis latrans), and the gray wolf (Canis lupus) are its definitive hosts that can excrete environmentally resistant oocysts in the environment, but also can act as intermediate hosts, harboring tissue stages of the parasite. In an attempt to isolate viable N. caninum from tissues of naturally infected wolves, brain and heart tissue from 109 wolves from Minnesota were bioassayed in mice. Viable N. caninum (NcWolfMn1, NcWolfMn2) was isolated from the brains of two wolves by bioassays in interferon gamma gene knockout mice. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates were analyzed by N. caninum-specific Nc5 polymerase chain reaction and confirmed diagnosis. This is the first report of isolation of N. caninum from tissues of any wild canid host.

Veterinary Parasitology, Nov 1, 2013

The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondi... more The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondii in freerange chickens from Grenada, West Indies. Using the modified agglutination test, antibodies to T. gondii were found in 39 (26.9%) of 145 free-range chickens with titers of 25 in 7 chickens, 50 in 6 chickens, 100 in 2 chickens, and 200 or higher in 24 chickens. The hearts of the 39 seropositive chickens were bioassayed in mice; viable T. gondii was isolated from 20 and further propagated in cell culture. Genotyping of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed 4 genotypes, including ToxoDB PCR-RFLP no. 2 (Type III), no. 7, no. 13, and no. 259 (new). These results indicated that T. gondii population genetics in free-range chickens seems to be moderately diverse with ToxoDB no. 2 (Type III) as the most frequent (15/20 ¼ 75%) compared to other genotypes in Grenada.

Journal of Parasitology, Oct 1, 2013

The ingestion of uncooked infected white-tailed deer (WTD) tissues can transmit Toxoplasma gondii... more The ingestion of uncooked infected white-tailed deer (WTD) tissues can transmit Toxoplasma gondii infection to humans and mesocarnivores, including cats. In the present study, we tested 264 WTD from New Jersey for T. gondii infection during the 2011-2012 hunting season. Serum samples were tested for antibodies to T. gondii by the modified agglutination test (cutoff titer, 25); 76 (28.7%) of 264 WTD were seropositive. Heart muscle samples from 64 seropositive WTD were digested in pepsin, and the digests were bioassayed for the isolation of T. gondii . Viable T. gondii was isolated in mice from the myocardium of 9 WTD; tachyzoites from infected mouse tissues were further propagated in cell culture. One of the 9 strains was highly virulent for outbred Swiss Webster mice. The DNA isolated from culture-derived tachyzoites of these 9 T. gondii isolates was characterized using 11 PCR-RFLP markers (SAG1, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Six genotypes were found, including ToxoDB genotype no. 2 (Type III), no. 3 (Type II variant), no. 4 (Type 12), no. 216, no. 220, and no. 221. The last 2 were new genotypes that were reported for the first time. This is the first report of T. gondii infection in deer from this region of the United States.

Veterinary Parasitology, Nov 1, 2013

Nothing is known of the genetic diversity of Toxoplasma gondii circulating in wildlife in Mexico.... more Nothing is known of the genetic diversity of Toxoplasma gondii circulating in wildlife in Mexico. In the present study, a mouse virulent T. gondii strain was isolated from the heart of a wild puma (Felis concolor). The puma was found roaming in outskirt of Durango City, Mexico and tranquilized for moving to a zoo. The puma died during translocation and a necropsy examination was performed. The puma had an antibody titer for T. gondii of 200 by the modified agglutination test. Its heart and brain tissue were bioassayed into 2 outbred Swiss Webster (SW) and 1 gamma interferon gene knockout (KO) mouse. The KO mouse and the 2 SW mice that became infected after inoculation with homogenate of puma heart died of acute toxoplasmosis 12, 19 and 20 days p.i. respectively and tachyzoites were found in lungs of all 3 mice. None of the 4 SW and 1 KO mouse inoculated with digest of the puma brain became infected with T. gondii. Tachyzoites from the lungs of mice were propagated in cell cultures. Tachyzoites from cell culture were inoculated into 5 SW; the mice died or had to be killed 14 days p.i. and a cat fed tissues of these mice shed T. gondii oocysts. Results of mortality and infectivity of tachyzoites and oocysts in SW mice indicated that the puma T. gondii strain (designated TgPumaMe1) was virulent for outbred mice. DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers (SAG1, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed a new genotype (ToxoDB PCR-RFLP #222). Isolation of atypical genotype T. gondii from wild puma indicates that mouse virulent strains are circulating in wildlife in Mexico.

Veterinary Parasitology, 2014

Donkeys (Equus asinus) are used as both companion and working animals throughout the world and in... more Donkeys (Equus asinus) are used as both companion and working animals throughout the world and in some countries, their meat and milk are used for human consumption. Here we report the first serological survey of Toxoplasma gondii in donkeys in the United States. Serum samples from 373 donkeys from eight farms in five states were tested for T. gondii antibodies by the modified agglutination test (MAT). Twenty-four of 373 (6.4%) of donkeys were seropositive, with MAT titers ranging from 25 to ≥ 200. All seropositive donkeys were Miniature breed. Seropositivity prevalence was 7.0% in female donkeys (20/282) and 4.1% in male donkeys (4/91). No donkeys less than 24 months of age (129) were seropositive, suggesting postnatal transmission of infection. Domestic cats were present on six of the eight farms. Three cats from one farm had MAT titers of 200. Viable T. gondii was isolated from the hearts of two cats, but not from brain tissues. Genotyping of isolate DNA extracted from culture-derived tachyzoites using 10 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico loci) revealed that both isolates were clonal Type II (ToxoDB PCR-RFLP genotype #1). This is the first serological survey for T. gondii in donkeys in the United States, and suggests that donkey milk and meat should be considered as a potential source for human infection. The role of barn cats in the transmission of T. gondii to donkeys on farms warrents further investigation.

Veterinary Parasitology, Feb 1, 2014

Recent studies indicated the predominance of Toxoplasma gondii haplogroup 12 in wildlife in USA. ... more Recent studies indicated the predominance of Toxoplasma gondii haplogroup 12 in wildlife in USA. But still little is known of the genetic diversity of this parasite circulating in wildlife. In the present study, we tested coyotes (Canis latrans), red foxes (Vulpes vulpes), white-tailed deer (Odocoileus virginianus), and geese (Branta canadensis) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 160 of 367 animals, including 92 (34.5%) of 266 coyotes, 49 (62.0%) of 79 white-tailed deer, 17 (85.0%) of 20 red fox, and two of two Canada geese tested by the modified agglutination test (cut off titer 1:25). Tissues from 105 seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 29 animals, including 10 of 53 coyotes, 11 of 16 foxes, 7 of 49 deer, and one of one goose. DNA isolated from culture-derived tachyzoites of these isolates was characterized initially using multilocus PCR-RFLP markers. Nine genotypes were revealed, including ToxoDB PCR-RFLP #1 (4 isolates), #2 (2 isolates), #3 (4 isolates), #4 (6 isolates), #5 (4 isolates), #54 (1 isolate), #141 (1 isolate), #143 (1 isolate), and #216 (6 isolates), indicating high genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of six T. gondii isolates (5 of #216 and #141) was determined in outbred Swiss Webster mice. Three of #216 and the #141 isolates were acute virulent to mice, and the other 2 #216 isolates were intermediate virulent. To determine the extent of genetic variation of these as well as a few recently reported virulent isolates from wildlife in North America, intron sequences were generated. Analysis of intron sequences and PCR-RFLP genotyping results *

Veterinary Parasitology, Sep 1, 2013

Recent studies indicate greater genetic variability among isolates of Toxoplasma gondii worldwide... more Recent studies indicate greater genetic variability among isolates of Toxoplasma gondii worldwide than previously thought. However, there is no information on genetic diversity of T. gondii from any host in Ethiopia. In the present study, genotyping was performed on viable T. gondii isolates by bioassays in mice from tissues and feces of 27 cats from Ethiopia. Viable T. gondii was isolated from hearts of 26 cats, feces alone of 1 cat, and feces and tissues of 6 cats; in total there were 33 isolates. Genotyping was performed on DNA from cell-cultured derived T. gondii tachyzoites and by using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Four genotypes were recognized, including ToxoDB #1 (Type II clonal, nine isolates), ToxoDB #2 (Type III, five isolates), Toxo DB #3 (Type II variant, ten isolates), and ToxoDB #20 (nine isolates). Of interest is the isolation of different genotypes from tissues and feces of two cats, suggesting re-infection or mixed strain T. gondii infection. These findings are of epidemiological significance with respect to shedding of oocysts by cats. This is the first report of genotyping of T. gondii from any host in Ethiopia.

Journal of Parasitology, Aug 1, 2013

Journal of Parasitology, Feb 1, 2013

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that ... more Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that excrete environmentally resistant oocysts in feces. In the present study, hearts, serum, and feces from 36 feral cats from Addis Ababa area, Ethiopia, were examined for T. gondii infection. Antibodies to T. gondii were determined with the modified agglutination test (MAT, cutoff 1:25); 33 cats were seropositive. Hearts of all 36 cats were homogenized, digested in pepsin, and bioassayed in mice. Feces were examined for T. gondii oocysts by bioassay in mice. Viable T. gondii was isolated from heart of 26 by bioassay in mice and from 25 seropositive and 1 seronegative cats. Toxoplasma gondii was isolated from feces (oocysts) by bioassay in mice. In total, viable T. gondii was isolated from 27 of the 36 cats, and these isolates were designated TgCatEt1 to TgCatEt27. The high prevalence of T. gondii oocysts in feces of 8 (19.4%) of 36 cats is of high epidemiologic significance. This is the first report of isolation of viable T. gondii from any host in Ethiopia.

Veterinary Parasitology, Nov 1, 2013

The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondi... more The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondii in freerange chickens from Grenada, West Indies. Using the modified agglutination test, antibodies to T. gondii were found in 39 (26.9%) of 145 free-range chickens with titers of 25 in 7 chickens, 50 in 6 chickens, 100 in 2 chickens, and 200 or higher in 24 chickens. The hearts of the 39 seropositive chickens were bioassayed in mice; viable T. gondii was isolated from 20 and further propagated in cell culture. Genotyping of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed 4 genotypes, including ToxoDB PCR-RFLP no. 2 (Type III), no. 7, no. 13, and no. 259 (new). These results indicated that T. gondii population genetics in free-range chickens seems to be moderately diverse with ToxoDB no. 2 (Type III) as the most frequent (15/20 ¼ 75%) compared to other genotypes in Grenada.

Veterinary Parasitology, 2013

Stray dogs are considered as sentinels in the epidemiology of Toxoplasma gondii because they are ... more Stray dogs are considered as sentinels in the epidemiology of Toxoplasma gondii because they are carnivores and eat variety of foods, including garbage. In the present study, tissues and sera of 51 stray dogs (Canis familiaris) from Giza, Egypt were examined for T. gondii infection. Sera were examined for antibodies to T. gondii by the modified agglutination test (MAT); 50 of 51 (98%) were seropositive with titers of 20 in four, 40 in four, 80 in one, 100 in eight, 200 in 17, 400 in 11, 800 or higher in five. Hearts of 43 seropositive dogs were bioassayed in mice. Viable T. gondii was isolated from 22 dogs; these isolates were designated TgDogEg1 to TgDogEg22. DNA isolated from cell culture derived tachyzoites of 22 isolates was genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed three genotypes and one mixed infection. The three genotypes are ToxoDB PCR-RFLP #2 (type III, four isolates), #3 (type II variant, 11 isolates), #20 (six isolates), 1 mixed infection. These results revealed the dominance of clonal type II, III and ToxoDB #20 lineages of T. gondii in stray dogs from Giza, Egypt.

Veterinary Parasitology, 2011

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. N... more The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum.

Journal of Parasitology, 2007

Two independently derived, exotoxin A-deficient (Tox-phenotype), nitrosoguanidine-induced mutants... more Two independently derived, exotoxin A-deficient (Tox-phenotype), nitrosoguanidine-induced mutants of Pseudomonas aeruginosa PAO 1 were isolated by using sensitive immunological assays. One mutant, designated PAOT1O, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT1O and the parental strain. The mutation in PAOT1O (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.

International Journal for Parasitology, 2011

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the pre... more Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5 0-and 3 0-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types.

Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 2012

Journal of Zoo and Wildlife Medicine, Dec 31, 2013

Little is known of the genetic diversity and epidemiology of Toxoplasma gondii infection in wildl... more Little is known of the genetic diversity and epidemiology of Toxoplasma gondii infection in wildlife in Caribbean Islands. The prevalence and genetic diversity of T. gondii in mongooses (Herpestes auropunctatus) was investigated. During 2011 and 2012, 91 mongooses were trapped in different parts of Grenada, bled, euthanized, and examined at necropsy. Antibodies to T. gondii were found in 27 mongooses tested by the modified agglutination test (cut-off titer 25). Muscles (heart, tongue, neck) of 25 of the seropositive mongooses were bioassayed for T. gondii infection in mice. Viable T. gondii was isolated by bioassay in mice from four mongooses with MAT titers of 1:50 in two, 1:200 for one, and 1:400 for one mongoose. The four T. gondii isolates were further propagated in cell culture. Strain typing of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed one isolate belongs to the Type III (ToxoDB #2) lineage, two to ToxoDB#7 lineage, and one to the ToxoDB #216 lineage. This is the first report of T. gondii isolation and genotyping in H. auropunctatus worldwide.