Modified Direct Agglutination Test Research Articles

Neospora caninum Infection in Cattle in the Province of Kohgiluyeh and Boyer Ahmad, Southwest of Iran: Seroprevalence and Molecular Assessment

Introduction Among the protozoa of veterinary importance, Neospora caninum is responsible for large economic and productive losses in cattle herds. Dogs are being considered the definitive hosts of the parasite. Neospora caninum has gained considerable attention through its role in the etiology of bovine abortion. The current study aimed at assessing the status of Neospora infection in cattle in Boyer-Ahmad County in Kohgiluyeh and Boyer-Ahmad province, southwest of Iran. Methods In this cross-sectional study, 150 cattle blood samples were collected and samples were screened for N. caninum antibodies using a modified direct agglutination test (MAT). For the same samples, 130 buffy coats were collected and tested, by PCR, for the presence of N. caninum DNA, targeting the Nc-5 gene. Results Anti-N. caninum antibodies were detected in the sera of 49 out of 150 cattle which is corresponding to a seroprevalence rate of 32.7%. Neospora DNA was detected in buffy coats of 26 out of 127 (20.47%) cattle. Even though Neospora infection was more common in females and in five-year-old cattle by serology and molecular methods, yet there was no statistically significant difference between age, sex, and Neospora infection in both molecular and serological methods (p > 0.05). Conclusion Findings of the current study indicate a high rate of N. caninum infection in cattle of Boyer-Ahmad region in the southwest of Iran. This issue should be further investigated and the prevention and control of this parasite in livestock, due to the high financial burden of this parasite in the livestock industry, should be considered.

A longitudinal study of Toxoplasma gondii seroconversion on four large Danish sow farms

Serological tests are routinely used to detect Toxoplasma gondii specific IgG antibodies in serum. Serological surveys of T. gondii show a medium to high prevalence in Danish indoor sows at the time of slaughter. However, little is known about when sows acquire T. gondii, and for how long they remain seropositive. Our focus was on quantifying the incidence rates in different age-cohorts and on investigating the T. gondii IgG antibody dynamics in sows. Four large Danish sow farms were longitudinally surveyed for 1 year. A total of 320 animals from 6, 12, 18, and 24-months age-cohorts were sampled at 5-week intervals. In total, 2989 plasma samples were tested using commercial enzyme linked immunosorbent assay (ELISA). The incidence rate in each of the four age-cohorts was calculated, and a time-to-event analysis was applied to the interval censored data to investigate the relationship between age and probability of T. gondii seroconversion. In the initial ELISA testing, eight sows tested positive for T. gondii at first survey, of which seven remained seropositive throughout the follow-up period. Additionally, 16 sows seroconverted, but only five of these remained seropositive. Weekly incidence rates in the 6, 12, 18, and 24-month age-cohorts were 0.0017 (95% CI = 0.0008 – 0.0037), 0.0009 (95% CI = 0.0003 – 0.0027), 0.0003 (95% CI = 0.0000 – 0.0018), and 0.0023 (95% CI = 0.0010 – 0.0051), respectively. Time-to-event analysis suggested that the incidence rate increased with age but could not conclude this definitively. The retesting of a subsample of the sows (n = 200) with the same ELISA and with modified direct agglutination test (MAT), and western blot (WB) assays suggested that 12 out of the 24 initial ELISA seropositive sows may have been false positive. These 12 sows also showed fluctuating antibody dynamics in ELISA. Hence, the unstable antibody dynamics in ELISA may pose a challenge for serological surveys of T. gondii in sows.

Polymerase chain reaction based detection of Toxoplasma gondii from lymph node aspirates of dogs

The purpose of the study was to detect DNA of Toxoplasma gondii in lymph node aspirates of dogs using amplification of SAG-2 gene fragment. Blood and lymph node aspirates were collected from 20 clinically ill dogs of different age groups. Lymph node aspirates were analysed with polymerase chain reaction (PCR). Two numbers of dogs were found to be positive by PCR. The PCR positive dogs showed modified direct agglutination test (MDAT) titre of 1:512 and 1:4,096. The remaining sera samples showed the titre value of less than 1:64 by MDAT and were also negative by PCR. This study indicates that lymph node aspirates could be an alternative ideal specimen for the detection of T. gondii.

Seroprevalence of Toxoplasma Gondii in Immunocompetent and Immunocompromised Patients Using IgG - Modified Direct Agglutination Test (IgG MAT)

Background/Objective:The present study was conducted to assess the seroprevalence of Toxoplasma gondiiin and around Tirunelveli, Tamil Nadu, India by in-house IgG assay, using MAT. Methods:Serum samples from 175 immunodeficient and 175 immunocompetent patients were collected at Tirunelveli District, Tamil Nadu from May 2006 to October 2007. They were subjected to in-house IgG assay using Modified Direct Agglutination test (MAT). Results:Out of 350 patients tested by MAT, 39 patients (11.41%) had antibodies for toxoplasmosis with mean MAT titre of 53.85 ± 55.28 and the titre ranged from 1:20 to 1:160. Among the immunocompetent group of 175 patients, 18 patients (10.29%) had antibodies to toxoplasmosis whereas in immunodeficient group of 175 patients, 21 patients (12%) had antibodies for toxoplasmosis. The sensitivity, specificity of IgG MAT in detecting toxoplasmosis was 80 and 90 % respectively. Statistical analysis: The results were analysed by using the statistical software SPSS. Conclusions:The overall seroprevalence of toxoplasmosis in and around Tirunelveli District of Tamil Nadu was 11.41% based on IgG MAT. The study has proved that MAT is recommended as the most convenient method for serodiagnosis of toxoplasmosis and has the additional advantage of not requiring species-specific conjugates.

Detection of Toxoplasma gondii DNA in the milk of naturally infected ewes

Toxoplasmosis is the major parasitic disease affecting sheep. It is important for veterinary medicine, animal science and public health since it causes reproductive and economic losses in the herd, as well as damaging human health due to consumption of contaminated meat and milk, which can facilitate zoonotic transmission. Detection of Toxoplasma gondii in ovine milk and lack of data in the literature describing differentiation between acute and chronic disease for this species stimulated the elaboration of the present research project. To achieve the aim of this study, the animals were allocated to two groups of 20 ewes each, of which group 1 was composed of animals with positive serology and group 2 with negative serology. Acute and chronic stages of the disease were differentiated by modified direct agglutination test (MAT), in which antigens were fixed with formalin (MAT-AF) and methanol (MAT-AM). The parasite was detected in milk by polymerase chain reaction (PCR), and the molecular identity of the amplified products was confirmed by sequencing. The serological results indicated that sheep had a chronic infection profile. T. gondii DNA was detected in seven milk samples from five seropositive sheep, and twice in milk of two sheep. Sequences of species shared 97–100% identity with T. gondii. These findings allowed the hypothesis that the peripartum period may also lead to the resurgence of tissue T. gondii tachyzoites cysts which can circulate again and be excreted in the milk. This study used sheep naturally infected with T. gondii as a prerequisite for further investigations on the possible participation of this species in toxoplasmosis epidemiology and as a potential transmission route related to consumption of milk from infected sheep.

Seroprevalence of <i>Toxoplasma gondii</i> in Nazareth Town, Ethiopia

To determine seroprevalence of toxoplasmosis, assess its zoonotic importance and identify factors associated with seroprevalence. Questionnaire survey and serological studies. Questionnaire survey and 65 serum samples from male and female urban and peri-urban residents aged between 15 days and 65 years. Feeding habits, purpose of keeping cats and association with family members, serologic evidence of toxoplasmosis by the Modified Direct Agglutination Test (MDAT), examination of HIV status using the HIV-Spot Test. Over 86% of the interviewed people had a history of consumption of raw or undercooked mutton and had close contact with cats. Of the serum samples analyzed by the MDAT 60% had serologic evidence of Toxoplasma infection. Significantly higher MDAT tiers were encountered both in pregnant and immunocompromised individuals. The risk factors associated with Toxoplasma infection, i.e. raw or undercooked mutton consumption and presence of cats appeared significant. The significance of toxoplasmosis as a disease of zoonotic importance was demonstrated. Close contact between family members and the consumption of raw or undercooked mutton were the major risk factors in the transmission of the disease. Considering the relatively high prevalence as revealed by this study it would be important to conduct studies on a wider scale. It would also be important to increase public awareness and upgrade the knowledge on congenital toxoplasmosis.

Prevalence of Antibodies to Trypanosoma cruzi, Toxoplasma gondii, Encephalitozoon cuniculi, Sarcocystis neurona, Besnoitia darlingi, and Neospora caninum in North American Opossums, Didelphis virginiana, from Southern Louisiana

We examined the prevalence of antibodies to zoonotic protozoan parasites ( Trypanosoma cruzi, Toxoplasma gondii, and Encephalitozoon cuniculi) and protozoans of veterinary importance ( Neospora caninum, Sarcocystis neurona, and Besnoitia darlingi) in a population of North American opossums ( Didelphis virginiana) from Louisiana. Samples from 30 opossums were collected as part of a survey for T. cruzi in Louisiana. Frozen sera from these 30 opossums were examined using an indirect immunofluorescent antibody test (IFAT) against in vitro-produced antigenic stages of these protozoans. Additionally, 24 of the 30 samples were examined using hemoculture, and all 30 were examined in the modified direct agglutination test (MAT) for antibodies to To. gondii. The prevalences of reactive IFAT samples were as follows: 60% for T. cruzi, 27% for To. gondii, 23% for E. cuniculi, 17% for S. neurona, 47% for B. darlingi, and 0% for N. caninum. Hemoculture revealed that 16 (67%) of 24 samples were positive for T. cruzi, compared to 18 of 30 (60%) by IFAT. The sensitivity and specificity for the IFAT compared to hemoculture was 100% for each. The modified direct agglutination test revealed that 9 (30%) of the 30 samples from opossums had antibodies to To. gondii , compared to 8 (27%) using the IFAT. The sensitivity and specificity of the IFAT compared to the MAT was 100% and 72%, respectively.

Serological survey of caprine toxoplasmosis in Ethiopia: prevalence and risk factors

The study was conducted to determine the prevalence and risk factors of toxoplasmosis in goats in Southern and central Ethiopia between October 2005 and May 2006. A total of 641 goats sera were tested using Modified Direct Agglutination Test (MAT), of which 480 (74.8% CI: 71.3, 78.2) were found to be positive. The highest prevalence was recorded in South Omo zone (82%) while the lowest was observed in East Shewa zone (62.2%). The study revealed that goats raised in southern Ethiopia are at a greater risk of acquiring T. gondii infection (OR = 2.55, CI: 1.726, 3.776; p = 0.000) than those which are raised in central Ethiopia. The prevalence of anti T. gondii antibody was significantly higher in older goats than in kids (OR = 2.33, CI: 1.490, 3.655; p < 0.0002) and in females than in males (p < 0.0007; OR = 0.68, CI: 0.542, 0.849). No significant difference was observed among goats kept under various husbandry practices. The high prevalence of toxoplasmosis in Ethiopian goats suggests a high risk of human infections. Further epidemiological investigation, isolation and genotyping of T. gondii are planned.

Prevalence of Agglutinating Antibodies to Toxoplasma gondii in Adult and Fetal Mule Deer (Odocoileus hemionus) From Nebraska

Toxoplasma gondii is an apicomplexan parasite of mammals and birds. Herbivores acquire postnatal infection by ingesting oocysts from contaminated food or water. Toxoplasma gondii infection is common in white-tailed deer, Odocoileus virginianus, but little is known about the prevalence of infection in mule deer, O. hemionus. We examined sera from 89 mule deer from Nebraska for agglutinating antibodies to T. gondii using the modified direct agglutination test (MAT) with formalin-fixed tachyzoites as antigen. Thirty-one (35%) of the samples were positive at dilutions of > or = 1:25. Samples were examined from 29 fetuses from these mule deer and none were positive in the MAT. Sera from 14 white-tailed deer from Nebraska were also examined and 6 (43%) were positive for T. gondii. Samples were examined from 5 fetuses from these white-tailed deer and none was positive in the MAT. Our results in both deer species from Nebraska are similar to studies conducted in white-tailed deer from other regions of the United States. Our findings indicate that mule deer are frequently infected with T. gondii and that mule-deer meat may be a source of human infection.

Evaluation of the performance of the modified direct agglutination test (MAT) for detection of Toxoplasma gondii antibodies in dogs

Toxoplasmosis is a zoonosis that has been the subject of study in Brazil and worldwide. The dogs are sentinels for the infection and can carry Toxoplasma gondii in the environment. Seroepidemiological surveys of these animals are an important tool in the surveillance and control of the disease and inform decision-making in health programs. In this study the performance of the Modified Agglutination Test (MAT) in the serodiagnosis of canine toxoplasmosis is evaluated and compared to the indirect immunofluorescent-antibody test (IFAT). A sample of 157 dog sera from the county of Monte Negro, Rondonia, with 76.40% positive reactions for Toxoplasma gondii (IFAT =16) was analyzed using the MAT (=25), presenting sensitivity of 85.00% (Confidence Interval 95.00%: 79.4 - 90.60%) and specificity of 100.00%.

Serologic prevalence of Toxoplasma gondii in horses slaughtered for food in North America

Serum samples from 1788 horses slaughtered for food in North America were tested for antibodies to Toxoplasma gondii using the modified direct agglutination test (MAT). Antibodies to T. gondii were found by the MAT in 124 (6.9%) of 1788 sera; the titers were 1 : 20 (69 horses), 1 : 40 (37 horses), 1 : 80 (9 horses), and ≥1 : 160 (9 horses). A total of 339 selected horses were also tested by the Sabin–Feldman dye test (DT). Dye test antibodies were found in 54 horses with titers of 1 : 10 (29 horses), 1 : 20 (12 horses), 1 : 40 (4 horses) and 1 : 80 (9 horses). There was no correlation between the DT and the MAT.

Toxoplasma gondii Antibodies in Naturally Exposed Wild Coyotes, Red Foxes, and Gray Foxes and Serologic Diagnosis of Toxoplasmosis in Red Foxes Fed T. gondii Oocysts and Tissue Cysts

Antibodies to Toxoplasma gondii were determined in sera from 222 coyotes (Canis latrans), 283 red foxes (Vulpes vulpes), and 97 gray foxes (Urocyon cinereoargenteus) from Indiana, Kentucky, Michigan, and Ohio during 1990-1993. Sera were examined in 1:25, 1:100, and 1:500 dilutions by the modified direct agglutination test (MAT) with formalinized whole tachyzoites plus mercaptoethanol. Antibodies were found in 131 (59.0%) of 222 coyotes, 243 (85.9%) of 283 red foxes, and 73 (75.3%) of 97 gray foxes. Antibodies were also measured by different serologic tests in 4 littermate T. gondii-free red foxes fed T. gondii tissue cysts or oocysts; the fifth littermate fox was not fed T. gondii. Antibodies were measured in fox sera obtained 0, 14, and 36-55 days after infection with T. gondii. All 4 foxes fed T. gondii developed MAT and dye test antibody titers of 1:200 or more 14 days later. The latex agglutination test (LAT) and indirect hemagglutination test (IHAT) were less sensitive than MAT for the diagnosis of T. gondii infection in foxes. Antibodies were not detected by LAT (titer 1:64) in the 2 foxes fed tissue cysts nor by IHAT in 1 of the foxes fed tissue cysts. Toxoplasma gondii was isolated by bioassay in mice from tissues of all 4 foxes fed T. gondii. The control fox had no T. gondii antibodies detectable by any of the serologic tests.

Seroprevalence of antibodies against Toxoplasma gondii in free-ranging mammals in Iowa.

Serum samples from raccoons (Procyon lotor), striped skunks (Mephitis mephitis), Virginia opossums (Didelphis virginiana), and free-ranging house cats trapped in Iowa between 1984 and 1988 were tested for antibodies against Toxoplasma gondii using the modified direct agglutination test (MAT). Antibody titers > or = 1:32 were considered indicative of infection. Prevalence rates by species were estimated for raccoons at 134/885 (15%), skunks at 38/81 (47%), opossums at 12/53 (23%), and cats at 16/20 (80%).

Experimental Toxoplasma gondii Infection in Raccoons (Procyon lotor)

Six raccoons (Procyon lotor) without detectable Toxoplasma gondii antibodies were used. Four raccoons were inoculated orally (2 with oocysts and 2 with tissue cysts) with ME49 strain of T. gondii and 2 raccoons were not inoculated with T. gondii. All raccoons remained clinically normal. Raccoons were killed between 59 and 61 days after inoculation and portions of their heart, skeletal muscle, and brain were digested in pepsin solution, and homogenates were bioassayed in mice. Toxoplasma gondii was isolated from all 4 inoculated raccoons; from the heart of 3, skeletal muscles of 2 and the brain of none. All 4 inoculated raccoons developed antibody titers > or = 1:1,600 in the modified direct agglutination test (MAT) using whole formalinized tachyzoites. Toxoplasma gondii antibody titers of the raccoons not inoculated with T. gondii remained < 1:25, and T. gondii was not isolated from their tissues. It was concluded that muscle tissue from multiple sites including the heart was the tissue of choice for conducting parasitologic surveys for T. gondii in raccoons. Evaluation of the sera of the experimentally infected raccoons in the Sabin-Feldman dye test, latex agglutination test, and the indirect hemagglutination tests indicated that the MAT detected antibodies faster and in higher titers than did the other serological tests.

Comparison of Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent Antibody Test, and Direct Agglutination Test for Detecting Toxoplasma Gondii Antibodies in Naturally Aborted Ovine Fetuses

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to greater than 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.