Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.     

散养鸡新孢子虫、弓形虫血清与分子流行病学调查及风险因素分析

通过调查河南省散养鸡新孢子虫病和弓形虫病的血清与分子流行病学情况,评估该地区土壤受新孢子虫和弓形虫污染的程度。分别采用IFAT和MAT方法检测散养鸡血清中的新孢子虫和弓形虫的抗体,PCR方法检测部分血清阳性样品消化液的弓形虫抗原。结果发现,新孢子虫病的阳性率为23.26%(150/642;滴度1∶25;CI 95%=20.25~26.79),弓形虫的感染率为18.86%(132/700;滴度1∶25;CI 95%=16.13~21.93)。PCR检测弓形虫的结果为16%(4/25)。弓形虫感染与散养鸡的性别有关,公鸡感染率显著高于母鸡感染率(P<0.05)。结果表明,散养鸡的新孢子虫病和弓形虫病的感染率均较高,该地区人畜面临着较高的新孢子虫和弓形虫卵囊污染的风险,公共卫生安全形势严峻。散养鸡的新孢子虫病流行情况在国内首次报道。     

Genotyping of Toxoplasma gondii isolates from chickens from India

The present study was undertaken to isolate and genotype Toxoplasma gondii from free-range chickens (Gallus domesticus) from villages in Maharashtra and Tamil Nadu states of central and south India, respectively. Blood, heart, and brain from a total of 741 chickens were examined for T. gondii infection. Antibodies to T. gondii, as assayed with the modified agglutination test (MAT >or = 1:5) were found in 133 (17.9%) chickens. Hearts and brains of 186 chickens were bioassayed in mice. Additionally, hearts and/or brains of most of the seronegative (MAT < 1:5) chickens were fed to 20 T. gondii-free cats, while 32 seropositive chickens (MAT 1:5) were fed to 3 cats. T. gondii was not isolated from any of the chickens by mouse bioassay. Five of the cats that were fed seronegative chickens shed oocysts, while isolates were not obtained from any of the other cats fed seropositive chickens. These five isolates, along with the two that were previously isolated in India through cat bioassay, were genetically analyzed. Genotyping using the SAG 2 locus indicated that two isolates were type II and five were type III. Microsatellite analysis revealed allelic differences between and within the lineages. This is the first report of genetic characterization of any T. gondii isolate from India.     

Genetic diversity of Toxoplasma gondii isolates from chickens from Brazil

Until recently, Toxoplasma gondii was considered clonal with very little genetic variability. Recent studies indicate that T. gondii isolates from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. In the present study, we retyped 151 free range chicken isolates from Brazil including 117 newly isolated samples from 11 geographically areas (Alagoas, Bahia, Ceará, Maranh?o, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Norte, S?o Paulo, Sergipe, and Rondonia) and 34 previously reported isolates from the very north (Pará) and the very south (Rio Grande do Sul). Ten PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico were used to genotype all isolates. Overall analysis of 151 T. gondii isolates revealed 58 genotypes. Half (29/58) of these genotypes had single isolate and the other half of the genotypes were characterized with two or more isolates. Only 1 of 151 isolates was clonal Type I strain and 5 were clonal Type III strains. Two isolates had mixed infections. Clonal Type II strain was absent. One strain was Type II at all loci, except BTUB. The results confirm high genetic diversity of T. gondii isolates from Brazil.     

Toxoplasma gondii infection in sentinel and free-range chickens from Argentina

This study aimed at isolating and genotyping Toxoplasma gondii from serologically positive free-range chickens from Argentina, and to evaluate the use of sentinel animals during a short time period of exposure to determine environmental contamination with T. gondii oocysts. Two groups of chickens on six farms were compared in this study: (i) young, 2-3 month-old broiler-type chickens reared as sentinel animals on the farms and (ii) adult chickens reared on the same farms for more than one year. Seroconversion rates of 7.0% or 5.7% were observed in sentinel broiler chickens reared for a period of 74 days (January-April 2010) or 88 days (August-November 2010) respectively, as shown by a T. gondii specific immunofluorescent antibody test. Fifty-three percent (17 of 32) of adult chickens were positive and showed higher titres than sentinel animals. Isolation of T. gondii from tissues (brain and heart) of serologically positive chickens was achieved from six of seven free-range adult birds with IFAT titres of 200 and higher. The isolated parasites were analysed by multi-locus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The isolated T. gondii showed three different genotypes: two genotypes consisted in atypical allele combinations, and the remaining genotype had exclusively clonal type II alleles. All isolates obtained at a single farm, corresponded to the same genotype. The T. gondii genotypes observed are identical to those described in cats, dogs, chickens and capybaras elsewhere in South America. Two isolates, which showed different allele combinations in PCR-RFLP, were characterized in a mouse virulence assay. While one isolate showed a low virulence a second isolate was of intermediate virulence to mice.     

猫源弓形虫虫株的分离与鉴定

将猫的心、脑、舌用盐酸-胃蛋白酶溶液消化处理后接种小白鼠，经连续传代分离弓形虫虫株，用特异PCR方法对所分离的虫株进行了鉴定。分别从24只猫的样品中分离出8株弓形虫虫株，用弓形虫种特异的PCR方法对8个虫株进行鉴定。均得到弓形虫的特异条带；测序证明所扩增出的DNA片段确为弓形虫的核糖体DNA第一 内转录间隔区（ITS1）部分序列。研究结果表明，将动物组织用盐酸-胃蛋白酶溶液消化处理后接种小白鼠是一种分离弓形虫虫株较为理想的方法．特异PCR方法能准确、快速地鉴定弓形虫虫株。     

Genetic and biologic characteristics of Toxoplasma gondii infections in free-range chickens from Austria

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in free-range chickens (Gallus domesticus) from 11 Bio-farms in Austria was determined. Antibodies to T. gondii assayed by the modified agglutination test (MAT) were found in 302 of 830 (36.3%) chickens with titers of 1:10 in 50, 1:20 in 69, 1:40 in 53, 1:80 in 40, 1:160 or higher in 90. Hearts of 218 chickens with MAT titers of 10 or higher were bioassayed individually in mice. Tissues from 1183 chickens were pooled and fed to 15, T. gondii-free cats. Feces of the cats were examined for oocysts; 11 cats shed T. gondii oocysts. T. gondii was isolated from 56 chickens by bioassay in mice. Thus, there were 67 isolates of T. gondii from these chickens. Genotyping of these 67 isolates using the SAG2 locus indicated that all 33 were Type II. Phenotypically and genetically these isolates were different from T. gondii isolates from Brazil. None of the isolates was virulent for mice. This is the first report of isolation of T. gondii from chickens from Austria.     

Characterization of Toxoplasma gondii isolates in free-range chickens from Chile, South America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 85 free-range chickens (Gallus domesticus) from Chile was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 47 of 85 (55.3.9%) chickens with titers of 1:5 in six, 1:10 in four, 1:20 in four 1: 40 in three, 1: 80 in nine, 1: 160 in four 1:320 in nine, and 1: 640 or higher in eight. Hearts and brains of 47 chickens with titers of 1:5 or higher were pooled for each chicken and bioassayed in mice. Tissues from 16 seronegative (MAT<1:5) chickens were pooled and fed to one T. gondii-free cat. Feces of the cat were examined for oocysts but none was found based on bioassay of fecal floats in mice. Hearts and brains from seven seronegative (<1:5) were pooled and bioassayed in mice; T. gondii was not isolated. T. gondii was isolated by bioassay in mice from 22 chickens with MAT titers of 1:20 or higher. Genotyping of these 22 isolates using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed three genotypes. Seventeen isolates had type II alleles and four isolates had type III alleles at all loci. One isolate contained the combination of type I and III alleles. This is the first report of genetic characterization of T. gondii isolates from Chile, South America.     

Isolation and characterization of Toxoplasma gondii strains from stray cats revealed a single genotype in Beijing, China

Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. This study was aimed to determine the seropositivity, distribution of genotypes and mouse virulence of T. gondii from stray cats in Beijing, China. A total of 64 serum samples, 23 feces and tissue samples were collected from stray cats in Beijing. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). 57.8% (37/64) of these stray cats had titers of 1:20 or higher and were considered positive with infection. T. gondii oocysts were not found in feces of the 23 cats. Tissues of 23 cats were bioassayed in mice and 11 T. gondii isolates were obtained. The genotype of these isolates were identified by 11 PCR-RFLP markers, including SAG1, (3'+5')SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico. Only one genotype was identified. This genotype, designated as ToxoDB genotype #9, was previously reported in cats, pigs and human from Guangdong and Gansu provinces in China and animals from a few other countries. To determine mouse virulence of this lineage of parasites, one isolate was randomly selected and inoculated into BABL/c mice, the result showed that it is intermediately virulent to mice. These results indicated that an atypical, intermediately virulent T. gondii lineage is widespread in China. The high seropositivity of T. gondii in stray cats posts potential risk of transmission of the parasite to human population in the region.     

Isolation and Identification of Toxoplasma gondii Isolates from Cats in Eryuan and Nujiang

The assay was aimed to isolate Toxoplasma gondii (T.gondii) strains from stray cat in Eryuan and Nujiang of Yunnan province.The cat tissues (heart,liver,lung and brain) were digested by acid pepsin solution,intraperitoneally inoculated in Kunming mice,passaged at least 3 generations,and followed by specific PCR amplification of partial B1 gene using species-specific primers.Three T.gondii isolates were isolated from 18 stray cats,PCR result showed that we got the specific target band,and the sequence result of the specific PCR product showed that it was ribosome B1 gene sequence of T.gondii. Homology comparison analysis showed that the isolates was 100.0% homology with T.gondii B1.The method of inoculation into the mice with the tissues that was digested by acid pepsin solution was an effective way to isolate T.gondii strain from animals,and the specific PCR assay was an accurate method for the rapid identification of T.gondii.     

Isolation and genetic characterization of Toxoplasma gondii from alpaca (Vicugna pacos) and sheep (Ovis aries)

Alpacas are important to the economy of several countries. Little is known of Toxoplasma gondii infection in alpacas worldwide. In the present study, T. gondii was isolated and genetically characterized from alpacas for the first time. Alpacas (n?=?16) and rams (n?=?12) pastured on a farm in Virginia, USA, were examined at necropsy. Antibodies to T. gondii were determined by the modified agglutination test (MAT, 1:25) and found in 6 of 16 alpacas with titers of 1:100 (2 alpaca), 1:400 (2 alpacas), 1:800 (1 alpaca), and 1:1,600 (1 alpaca), and 5 of 12 rams in titers of 1:50 in one, 1:400 in one, 1:800 in one, 1:1,600 in one, and 1:3,200 in one. Tissues of all 16 alpacas were bioassayed in mice or in cats. Muscles (heart, skeletal muscle) of nine alpacas with MAT titers of 1:25 were fed to T. gondii-free cats; the cats did not shed oocysts. Viable T. gondii was isolated from tissues of two of six seropositive alpacas by bioassay in mice. Viable T. gondii was isolated from three of three seropositive sheep by bioassay in mice. Genotyping using cell-cultured tachyzoites revealed four genotypes, including one for ToxoDB PCR-RFLP genotype #2 (type III), one for genotype #3 (type II variant), one for genotype #170, and two for a new genotype designated as ToxoDB PCR-RFLP genotype #230. Thus, four of the five T. gondii isolates in the present study belonged to different genotypes. These results indicate a higher genetic diversity among T. gondii isolates circulating in the USA than previously realized.     

洱源、怒江猫源弓形虫虫株的分离与鉴定

为了分离猫源弓形虫,本试验从云南洱源、怒江两地区捕捉18只野猫,取其心脏、肝脏、肺脏、脑组织用盐酸—胃蛋白酶溶液消化处理后,腹腔接种小白鼠,将分离到的弓形虫虫株至少传3代,用特异PCR方法对所分离的虫株进行鉴定。结果表明,从18只野猫的样品中分离出3株弓形虫虫株,用特异性引物对3株虫株进行PCR鉴定,均得到弓形虫的特异性目的条带,测序结果表明所扩增出的DNA片段确为弓形虫核糖体B1基因部分序列。同源性比对分析结果显示分离株与T.gondii B1的同源性为100.0%。将动物组织用盐酸—胃蛋白酶溶液消化处理后腹腔接种小白鼠是一种分离弓形虫虫株较理想的方法,对弓形虫B1基因进行特异性扩增,可以快速地鉴定弓形虫虫株。     

Genetic and biologic characterization of Toxoplasma gondii isolates of cats from China

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 34 cats from People's Republic of China. Antibodies to T. gondii were assayed by the modified agglutination test and found in 27 of 34 (79.4%) cats with titers of 1:40 in one, 1:80 in one, 1:160 in three, 1:320 in three, 1:640 in eight, and 1:1280 or higher in 11 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, and tongue) of 27 seropositive cats were pooled and bioassayed in mice (8 cats) or cats (19 cats). T. gondii was isolated from tissues of 17 of 27 seropositive cats. Genotyping of these 17 T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and a new SAG2, and an apicoplast marker Apico revealed two genotypes. This is the first report of genetic typing of T. gondii isolates from cats from China.     

Isolation of Toxoplasma gondii from goats with history of reproductive disorders and the prevalence of Toxoplasma and chlamydial antibodies

The prevalence of antibodies to Toxoplasma gondii and Chlamydia psittaci was assessed in goats with a history of abortion, stillbirth and neonatal mortality. Antibodies were detected in 540 (30%) and 57 (3.2%) goats out of 1799 tested by indirect haemagglutination and complement fixation tests, respectively. Toxoplasma gondii was isolated for the first time in Botswana from 22 out of 81 sets (27.2%) of foetal tissues, maternal and foetal cotyledons and uterine tissues of goats which had previously aborted or given birth to stillborn or weak kids that died within two days of birth. These results implicate T. gondii and C. psittaci, but especially the former, to be associated with caprine reproductive problems and require appropriate control measures.     

Genetic and biologic characteristics of Toxoplasma gondii isolates in free-range chickens from Colombia, South America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 77 free-range chickens (Gallus domesticus) from Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 32 (44.4%) of 72 chickens with titers of 1:5 in 4, 1:10 in 3, 1:20 in 1, 1:40 in 1, 1:80 in 8, 1:160 in 8, 1:320 in 3, and 1:640 or higher in 4. Hearts and brains of 31 seropositive chickens were pooled and bioassayed in mice. Tissues from 32 (16+16) seronegative chickens were pooled and fed to two, T. gondii-free cats, and tissues from nine chickens without matching sera were fed to one T. gondii-free cat. Feces of cats were examined for oocysts. T. gondii oocysts were excreted by a cat that was fed tissues of 16 seronegative chickens. T. gondii was isolated by bioassay in mice from 23 chickens with MAT titers of 1:20 or higher. All infected mice from 16 of the 23 isolates died of toxoplasmosis. Overall, 82 (81.1%) of 101 mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 24 isolates using polymorphisms at the SAG2 locus indicated that seven T. gondii isolates were Type I, 17 were Type III, and none was Type II. Phenotypically, T. gondii isolates from chickens from Colombia were similar to isolates from Brazil but different from the isolates from North America; most isolates from chickens from Brazil and Colombia were lethal for mice whereas isolates from North America did not kill inoculated mice. Genetically, none of the T. gondii isolates from Colombia and Brazil was SAG2 Type II, whereas most isolates from chickens from North America were Type II. This is the first report of genetic characterization of T. gondii isolates from Colombia, South America.     

Biologic and genetic characteristics of Toxoplasma gondii isolates in free-range chickens from Nicaragua, Central America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 98 free-range chickens (Gallus domesticus) from Nicragua was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 84 (85.7%) of 98 chickens with titers of 1:5 in 10, 1:10 in eight, 1:20 in seven, 1:40 in nine, 1:80 in 11, 1:160 in one, 1:200 in 27, 1:400 in six, 1:800 four, and 1:3200 in one bird. Hearts and brains of 32 chickens with titers of 1:10 or less were pooled and fed to three T. gondii-free cats. Hearts and brains of 66 chickens with titers of 1:20 or higher were bioassayed in mice. Feces of cats were examined for oocysts. The cat fed tissues from eight chickens with titers of 1:10 shed T. gondii oocysts. The two cats fed tissues of 24 chickens with titers of 1:5 or less did not shed oocysts. T. gondii was isolated by bioassay in mice from 47 chickens with MAT titers of 1:20 or higher. All infected mice from six isolates died of toxoplasmosis. Overall, 41 of 170 (24.1%) mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 48 isolates (47 from mice and 1 from pooled tissues) using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed eight genotypes. Six isolates had Type I alleles, three isolate had Type II alleles and six isolates had Type III alleles at all loci. Four isolates had mixed infections. Two isolates have a unique allele at SAG1 locus and combination of I and III alleles at other loci. The rest 27 isolates contained the combination of Type I and III alleles and were divided into four genotypes. More than one genotypes were often isolated in chickens from the same household, indicating multiple genotypes were circulating in the same environment. This may explain the high frequency of mixed infections observed. High rate of mixed infection in intermediate hosts such as chickens may facilitate genetic exchange between different parasite lineages in definitive feline hosts. This is the first report of genetic characterization of T. gondii isolates from Nicragua, Central America.     

Genotyping of Toxoplasma gondii isolates from free range chickens in the Pantanal area of Brazil

The aim of this paper was to genetically characterize Toxoplasma gondii isolates from free range chickens in regions of Brazilian territory in the state of Mato Grosso do Sul (MS) where T. gondii strains have never been studied. In total, T. gondii isolates from 22 free range chickens were included in this study. Fifty chickens from Eldorado, thirty from Rio Verde and ten from Aquidauana were sampled between January and April 2007. In relation to the genetic diversity of T. gondii isolates from chickens in MS, the magnitude of the diversity in the isolates sampled in this study was comparable to the overall diversity in a composite data set. These 22 isolates in MS revealed 11 genotypes, whereas the 321 isolates ever genotyped in Brazil have revealed 95 genotypes. The values of Simpson's Diversity Index for the whole population of T. gondii isolates in Brazil, the whole population of T. gondii isolates from chickens in Brazil and the population surveyed in this study were 0.97, 0.95 and 0.90, respectively. Seven of the 11 genotypes revealed from chicken isolates from MS are newly described genotypes and six of them each have a single isolate. In conclusion, the results obtained from isolates in MS corroborate previous studies on T. gondii isolates in Brazil, thus confirming their diversity and atypicality. Nonetheless, the applicability of PCR-RFLP markers for epidemiological inferences remains controversial.     

猪弓形虫病的诊断及虫体分离鉴定

江西某猪场的后备母猪发生以体温升高、呼吸困难、皮肤发绀为主要特征的疫病,剖检病死猪的病理变化主要表现为肺水肿,肺脏、肝脏和脾脏肿大并有少量灰白色坏死点,全身淋巴结肿大和出血。肺门淋巴结触片,经瑞氏染色镜检可见镰刀形或半月形的弓形虫滋养体;无菌取肺门淋巴结腹腔接种BALB/c鼠,分离得一株弓形虫,该弓形虫分离株对小鼠具有稳定的致病力,小鼠接种弓形虫腹水(0.05~0.1 mL/只)后4~5 d死亡,腹水中有大量成丛、双在或分散的弓形虫滋养体。根据流行病学、临床症状、病理变化及实验室检查结果,诊断该后备母猪场的疫病为猪弓形虫病。     

Isolation and characterization of Ornithobacterium rhinotracheale from chickens in Brazil

Ornithobacterium rhinotracheale (ORT) is a recently described species of bacterium associated with respiratory disease, growth retardation, mortality, and decreased egg production in chickens and turkeys. Pneumonia, pleuritis, and airsacculitis characterise the infection. ORT has been isolated in many countries but it is still considered exotic in Brazil. Up to date it is prohibited to import and produce reagents for diagnostic and vaccination control. The aim of this study was to isolate and identify the bacteria in chickens. Four isolates were obtained from tracheal swabs of broilers. They were isolated in blood agar with gentamicin and showed biochemical, morphological, antigenic and genetic characteristics of ORT. The results confirm that ORT is present in Brazil.