High seroprevalence of Toxoplasma gondii and probability of detecting tissue cysts in backyard laying hens compared with hens from large free-range farms

Serological assays are commonly used to determine the prevalence of Toxoplasma gondii infection in livestock, but the predictive value of seropositivity with respect to the presence of infective tissue cysts is less clear. The present study aimed at the identification of seropositive and seronegative free-range laying hens from organic and backyard farms, and the relationship with the presence of viable tissue cysts. In addition, potential risk and protective factors on the selected farms were investigated. An in-house T. gondii surface antigen (TgSAG1, p30, SRS29B) ELISA was validated with sera from experimentally infected chickens and used to examine 470 serum samples collected from laying hens from organic and backyard farms at the end of their laying period. A total of 11.7% (55/470) of all chickens tested positive, and another 18.9% (89/470) of test results were inconclusive. The highest seroprevalences were observed on small backyard farms with 47.7% (41/86) of chickens being seropositive while another 20.9% (18/86) of test results were inconclusive. Twenty-nine seropositive, 20 seronegative and 12 laying hens which yielded inconclusive ELISA results, were selected for further examination. Hearts and limb muscles of these hens were examined for T. gondii tissue cysts in a bioassay with IFNɣ-knockout or IFNɣ-receptor-knockout mice. Viable T. gondii was isolated from 75.9% (22/29) of the seropositive, 25.0% of the inconclusive (3/12), and 5.0% (1/20) of the seronegative chickens. All 26 chickens tested positive in heart samples, while drumstick muscles (i.e. limb muscles) tested positive only in three. Data on putative risk and protective factors were collected on the farms using a standard questionnaire. Generalised multilevel modelling revealed farm size, cat related factors (‘cats on the premise’, ‘cats used for rodent control’), hen house/hall related factors (‘size category of hen house/hall’, ‘frequency category of cleaning hen house/hall’, ‘service period’) as significantly associated with seropositivity to T. gondii in hens. The final model, which included the age of the birds as an effect modifier and farm as a random effect variable, revealed that the use of cats for rodent control and an area available per hen in the chicken run of >10 sqm were statistically significant risk factors for T. gondii seropositivity. Overall this study showed that exposure to T. gondii is common in small backyard farms but is rare on large organic farms with a high density of free-range hens, even when cats were present on the premises.