A novel luciferase-linked antibody capture assay (LACA) for the diagnosis of Toxoplasma gondii infection in chickens.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the most common parasitic zoonosis worldwide in multiples species of mammals and birds. Although free-range chickens may play a role as an important reservoir for T. gondii, there is no reliable and commercially available diagnostic test for this disease in chickens. In this study, we aimed to develop a novel Luciferase-linked Antibody Capture Assay (LACA) for the serodiagnosis of Toxoplasma infection in chickens. Recombinant nanoluciferase fused-T. gondii dense granule antigen 8 (rNluc-GRA8) was produced and applied to LACA assay as a diagnostic antigen. GRA8-LACA was tested with the sera from uninfected and experimentally infected chickens with T. gondii and other parasitic pathogens and showed unexpectedly high sensitivity (90.5%) and specificity (95.4%). Interestingly, E. coli lysate expressing rNluc-GRA8 could be applied in GRA8-LACA with 85.7% sensitivity and an increased specificity (96.9%) that gave better diagnostic performance compared to conventional ELISA. We applied our diagnostic system to examine 267 free-range chicken sera collected from 12 farms and 100 closed-house broiler chicken sera from local poultry abattoirs. The overall seroprevalence of toxoplasmosis in free-range chickens was 10.9% (95% CI: 10.6%-11.1%), while no positive case was found in broiler chickens. GRA8-LACA could be a useful diagnostic technique for T. gondii infection in chickens. The detection of T. gondii seropositive chickens in this study warns a potential risk of Toxoplasma transmission by the consumption of raw or undercooked chicken meat.

A Toxoplasma gondii strain isolated in Okinawa, Japan shows high virulence in Microminipigs.

Toxoplasma gondii strains have been isolated all over the world and their virulence has been examined mainly using laboratory mice. However, T. gondii differs in virulence depending on the host animal species. Therefore, to evaluate the virulence of each strain in domestic animals, it is necessary to examine using not only mice but also the concerned animals. We have shown that TgCatJpOk4, a T. gondii strain recently isolated in Okinawa, Japan, has a high virulence against laboratory mice, comparable to highest virulent RH strain in mice; however, the virulence to domestic animals remains unknown. In this study, we examined the virulence using the Microminipig. After infection, four out of five infected pigs showed severe clinical symptoms: inappetence, hypoactivity and tachypnea. Eventually, three out of the five infected pigs succumbed before the end of the observation. Among the three dead pigs, histological analysis revealed that interstitial pneumonia and spotty necrosis in the liver indicating that the TgCatJpOk4 strain has a high virulence not only in laboratory mice, but in pigs as well.

First Molecular detection of Theileria annulata in Bangladesh.

In South Asia, Theileria annulata is known to be less pathogenic to local breeds of Bos indicus cattle comparing to Bos taurus cattle and some of mix breeds between them. Seroepidemiological surveys have revealed high sero-prevalence of T. annulata in asymptomatic local breeds of cattle in Bangladesh. Therefore, these asymptomatic infection in local breeds can be infectious sources to more sensitive breeds. In this study, 59 bloods of cattle showing no symptom were screened by species specific PCRs for hemoto-protozoan parasites, to prove the existence of T. annulata parasite in asymptomatic cattle in Bangladesh. The T. annulata infection was confirmed along with other parasitic species, and this is the first report of T. annulata DNA detection in Bangladesh.

Native SAG1 in Toxoplasma gondii lysates is superior to recombinant SAG1 for serodiagnosis of T. gondii infections in chickens.

Toxoplasma gondii can infect almost all mammals and birds, including chickens. The aim of this study was to identify an appropriate immunogenic antigen for serodiagnosis of T. gondii infections in chickens. We examined serum samples from chickens that were intravenously or intraperitoneally infected with 106-108 tachyzoites of T. gondii strains PLK, RH, CTG, ME49 or TgCatJpGi1/TaJ using enzyme-linked immunosorbent assays (ELISAs), latex agglutination tests (LATs) and western blotting. Regardless of parasite strain or infection dose and route, the commercial LAT was positive for almost all sera collected 1 week post-infection. However, at 2 weeks post-infection, LATs were negative in the same birds. ELISAs using the Escherichia coli-produced recombinant T. gondii antigens SAG1 and GRA7 showed strong signals at 1-2 weeks post infection, but thereafter diminished for the majority of serum samples. In contrast, western blotting against crude tachyzoite antigens showed a persistent band up to 4 weeks post-infection. Sera from these chickens reacted much more strongly with SAG1 from crude tachyzoite antigens than with recombinant SAG1. Even in experimentally-infected birds whose parasite burdens in tissue were undetectable, sera still reacted with native SAG1. We tested sera from free-range chickens on a small farm in Ghana, Africa, using western blotting and found that the serum of one bird reacted with a single band of approximately 27 kDa, the putative molecular weight of SAG1. Thus we conclude that native SAG1, but not E. coli-produced recombinant SAG1, is suitable for serodiagnosis of T. gondii infections in chickens.

Atypical virulence in a type III Toxoplasma gondii strain isolated in Japan.

The virulence of a type III Toxoplasma gondii strain isolated in Japan and designated here as TgCatJpGi1/TaJ was examined in mice and micro minipigs in this study. Despite its type III genotype, oral or intraperitoneal inoculation of cysts from it resulted in severe virulence in C57BL/6J and BALB/c mice. In contrast, mice inoculated with a high dose of TgCatJpGi1/TaJ tachyzoites showed no obvious clinical signs of infection, and all of them survived for >21 days post-inoculation. Furthermore, no clinical signs of infection were seen when micro minipigs were inoculated with 900 cysts. Interestingly, our allelic type screening of the virulence-related rop5, rop16, rop17, and rop18 genes, as based on restriction fragment length polymorphism analysis (RFLP), revealed that the RFLP patterns for TgCatJpGi1/TaJ were identical to those from nonvirulent type III parasites. These results suggest that TgCatJpGi1/TaJ possesses an unknown virulence factor or factors.

Theileria annulata seroprevalence among different cattle breeds in Rajshahi Division, Bangladesh.

An epidemiological survey of Theileria annulata infection was undertaken in a cattle population in Rajshahi Division, Bangladesh. The local cattle breeds from the area (North Bengal Gray and Deshi) and crosses between the local breeds and Holstein cattle were predominantly screened. In total, 192 cattle serum samples were collected in two areas of Rajshahi Division, the Rajshahi District (n=147) and Natore District (n=45). The samples were screened with an enzyme-linked immunosorbent assay using T. annulata surface protein (TaSP) as the antigen. The seroprevalence was 80.0% (36/45) in Natore and 20.4% (30/147) in Rajshahi. A logistic regression analysis showed that the sampling location was significantly associated with seropositivity, whereas age, sex and breed were not. Although the logistic regression analysis did not show a linear dependence on age, we considered age-specific seroprevalence separately in the two districts. Seroprevalence did not differ significantly among age categories in the Natore District. In contrast, all the cattle <1 year old in the Rajshahi District were seronegative (11/11). Seroprevalence in the 1- and 2-year-old cattle was significantly lower in the Rajshahi District than in the Natore District. In the older age categories (3, 4 and >5 years), seroprevalence did not differ significantly between the Natore and Rajshahi Districts. These results suggest that the cattle in the Rajshahi District were sporadically exposed to T. annulata, whereas most cattle in the Natore District became infected during an early phase of life.