Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum

Abstract

dAlthough many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78. Coxiella burnetii causes Q fever in humans as well as abortions, stillbirths, and infertility in ruminants (1-5). The organism replicates in the placenta of infected ruminants, reaching a level of up to 10 9 bacteria per gram of placenta tissue (4-6). C. burnetii organisms are shed in an extremely high concentration in birth fluids, placental tissues, and membranes of aborted fetuses as well as in milk, urine, and feces of infected ruminant animals around the parturition period (4, 5). The high concentration of C. burnetii organisms shed in tissues, fluids, and excreta of infected ruminants is the primary source of human infections (7).Caprine and ovine infections have been reported to result in severe placentitis and consequently in shedding of higher numbers of C. burnetii organisms than infections of cattle (8). Studies have also revealed that goats and sheep shed higher quantities of C. burnetii in feces, vaginal mucus, and birth tissues than other livestock (9). Thus, the risk of human transmission is higher when infections occur in herds of small ruminants than when they occur with other livestock. Unsurprisingly, the majority of reported large outbreaks of Q fever have been associated with infected sheep and goat flocks, including a major outbreak of more than 4,000 human Q fever cases in the Netherlands that was linked to sheep and goat farms with over 50 animals (9)(10)(11)(12)(13)(14). Infection with C. burnetii can be asymptomatic in many animals and may be detected in ruminants only when infection causes abortions and reproductive abnormalities in pregnant animals (1). Delay in diagnosis in livestock slows the implementation of appropriate control strategies, thus increasing the risk of human infection.Coxiellosis in animals can be diagnosed through microscopic examination of stained tissues, culture, detection of C. burnetii DNA using PCR, and detection of antibodies to C. burnetii in blood and milk (15,16). The microscopic diagnosis of coxiellosis is mainly undertaken on placental tissues using Stamp-Macchiavello coloration or Giemsa stain. The organism can be cultured in cells, embryonated hen eggs, or cell-free media (15,17). However, microscopy and culture are expensive and requi...

Prevalence of Coxiella burnetii in German sheep flocks and evaluation of a novel approach to detect an infection via preputial swabs at herd-level

“…Results with values between 30% and 40% were considered inconclusive and were scored as negative in the present study. A sensitivity of 70.1%, 84% and 98.6% and a specificity of 96.2%, 99% and 97.1% were assumed according to Muleme et al, Paul et al and Horigan et al [21][22][23], taking into account that data were provided under different study designs and for different species.…”

“…The true mean proportion of infected adults detected by ELISA was 13% (95% CI 3-22) among 12 positive farms, 9% (95% CI 3-14) among 20 farms and 8% (95% CI 2-13) among 15 farms according to test quality specifications by Muleme et al, Paul et al and Horigan et al respectively[21][22][23]. Within 10 positive farms, 18% (95% CI 0-39) were detected by the qPCR.…”

Sero-Epidemiological Study of Selected Zoonotic and Abortifacient Pathogens in Cattle at a Wildlife-Livestock Interface in South Africa

“…April to September 2013, the location and management of cattle farms (Transvaal province vs. Mpumalanga province), and most importantly, serological tests used (Complement Fixation Test vs. ELISA). These factors have the potential to affect exposure and detection of C. burnetii (Muleme et al 2016, Plummer et al 2018, Larson et al 2019.…”

The prevalence of Coxiella burnetii shedding in dairy goats at the time of parturition in an endemically infected enterprise and associated milk yield losses

“…The com1 amplicon cloned into a plasmid and DNA extracted from Vero cell cultures of C. burnetii Nine Mile RSA439 (Phase II, Clone 4) were used as positive controls for the com1 and IS1111 assays, respectively. Serological testing was carried out using the methods described by [ 25 ] using a 1/160 dilution cut-off. For IFA negative controls serum from New Zealand goats (a country declared free of C. burnetii by the World Organisation for Animal Health) was used.…”

“…Compared with the com1 qPCR the IS1111 qPCR is more sensitive; an expected feature considering the insertion sequence it targets can occur up to 110 times within the C. burnetii genome [ 22 – 24 ]. Antibodies against C. burnetii can be assessed using a range of serological techniques of which the immunofluorescence assay (IFA) has the highest diagnostic sensitivity and specificity when used in goats [ 25 ].…”