Serological and Molecular Detection of Toxoplasma gondii in Domestic Pigs Intended for Human Consumption and Potential Occupational Hazard to Pig Farmers in India.

Toxoplasma gondii, an important food-borne zoonotic parasite, poses a worldwide public health hazard. Domestic pigs are considered one of the main intermediate hosts in the zoonotic transmission of T. gondii. To date, seroepidemiological information on T. gondii in domestic pigs in India is very scarce, and there are no reports of occupational hazards to pig farmers in this country. Here, we aimed at estimating the occurrence of T. gondii (antibodies and parasite DNA) in slaughtered pigs and pig farmers in Central India. Seroprevalence was determined in 410 serum samples from slaughtered pigs and 103 sera from pig farmers using an in-house prepared antigen-based modified agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and indirect-fluorescent antibody test (IFAT). Anti-T. gondii IgG antibodies were detected in 200 pigs (up to 48.8%, confidence interval [95% CI]: 40.4-52.2) and 44 pig farmers (up to 42.7%, 95% CI: 35.6-47.3) using MAT, ELISA, and IFAT. Inter-rater agreement showed an excellent agreement (kappa κ = 0.9) among the different serological tests suggesting similar detection potential of these tests. Recently acquired infections in all seropositive subjects were determined using IgG avidity testing and polymerase chain reaction (PCR). IgG avidity showed that 20 (10.3%) of slaughtered pigs and 8 (19.5%) pig farmers had a recently acquired infection. PCR for B1 and 529 repeats was performed in the heart tissues of slaughtered pigs and the blood cells of pig farmers. T. gondii DNA was detected in 14 (7.2%) slaughtered pigs and 5 (12.2%) pig farmers. Univariate analysis revealed that adult animals (>1 year), cats and rodents on the farm, and outdoor access are common factors (p ≤ 0.05) associated with T. gondii infection in pigs. Our results indicate that T. gondii is widely distributed in slaughtered pigs and pig farmers at risk of infection, highlighting a potential zoonotic transmission and health risk to consumers.

Seroprevalence, risk factors, and serological cross-reactivity for diagnosis of Toxoplasma gondii and Neospora caninum infections in goats in India.

Toxoplasma gondii and Neospora caninum are genetically related cyst-forming protozoan parasites that cause reproductive failures in ruminants. Given the limited information on the epidemiology of these infections in goats in India, the study aimed to estimate the seroprevalence, assess antibody cross-reactivity for diagnosis, and identify associated risk factors. A total of 695 sera were evaluated for antibodies to T. gondii and N. caninum infections using Modified Agglutination Test (MAT for Toxoplasma)/Neospora agglutination test (NAT), Enzyme-linked immunosorbent assay (ELISA), and Indirect Fluorescent Antibody Test (IFAT for tachyzoite and bradyzoite stages). The seroprevalence rate of T. gondii and N. caninum infections was 56.9% and 10.9%, respectively. Inter-rater agreement (kappa value - κ) was calculated to assess agreements between various diagnostic assays, using the IFAT as the gold standard (for detecting both infections), the agreements for MAT/NAT (κ = 0.97) and the ELISA (κ = 0.95) were excellent. The acute infection among seropositive goats were determined using serological (IgG avidity test - measures the binding strength between IgG antibodies and parasite antigens) and molecular diagnoses (PCR for repetitive DNA sequences - Toxoplasma B1 gene: 131 bp and Neospora NC5 gene: 328 bp). Among seropositive goats ≥80% had high IgG avidity and <10% of animals had low IgG avidity antibodies for both infections. Most low IgG avidity goats were PCR positive for the TgB1 gene (94.4%) or Nc5 gene (85.7%). In the serological assays, we used different dilutions of test serum to rule out the cross-reactivity owing to similar antigenic makeup between these two parasites. When the serological cross-reactivity was analyzed using invasion assay at a serum titer of ≥200, more than 90% T. gondii positive sera showed host cell invasion of N. caninum and vice versa. Largely, the serological results indicate that cut-off serum dilution of ≥1:200 for ELISA and IFAT and ≥1:25 for MAT/NAT avoids serological cross-reactivity between T. gondii and N. caninum. Further, the Univariate and multivariate analyses showed that adult animals (>2 years), reservoir hosts, and extensive rearing systems are common risk factors for these infections. However, the history of abortion was identified as a significant risk factor for T. gondii infection. This study revealed that T. gondii and N. caninum infections are highly prevalent in this region and the use of an appropriate cut-off serum dilution is necessary to avoid cross-reactivity between these closely related parasites.

Polymerization kinetics of tubulin from mung seedlings modeled as a competition between nucleation and GTP-hydrolysis rates.

Microtubules (MTs) form physiologically important cytoskeletal structures that are assembled by tubulin polymerization in nucleation- and guanosine triphosphate (GTP)-dependent manner. GTP hydrolysis competes with the addition of monomers, to determine the GTP-cap size, and the onset of shrinkage, which alternates with growth. Multiple theoretical models of MT polymerization dynamics have been reconciled to the kinetics of animal brain tubulins, but more recently, rapid kinetics seen in Arabidopsis tubulin polymerization suggest the need to sample a wider diversity in tubulin polymerization kinetics and reconcile it to theory. Here, we isolated tubulin from seedlings of Vigna sp. (mung bean), compared polymerization kinetics to animal brain tubulin, and used a computational model to understand the differences. We find that activity-isolated mung tubulin polymerizes in a nucleation-dependent manner, based on turbidimetry, qualitatively similar to brain tubulin, but with a 10-fold smaller critical concentration. GTP-dependent polymerization kinetics also appear to be transient, indicative of high rates of GTP hydrolysis. Computational modeling of tubulin nucleation and vectorial GTP hydrolysis to examine the effects of high nucleation and GTP-hydrolysis rates predicts a dominance of the latter in determining MT lengths and numbers. Microscopy of mung tubulin filaments stabilized by GMPCPP or taxol results in few and short MTs, compared to the many long MTs arising from goat tubulin, qualitatively matching the model predictions. We find GTP-hydrolysis outcompetes nucleation rates in determining MT lengths and numbers.