Comparison of conventional and molecular techniques for Trypanosoma vivax diagnosis in experimentally infected cattle

Abstract

Livestock infections by Trypanosoma vivax have been occurring with increasing frequency, mainly due to the presence of animals with subclinical infections and without apparent parasitaemia, making diagnosis challenging. The aim of the present study was to evaluate several techniques used for T. vivax diagnosis in order to assess the best way of using them during the course of the disease. Molecular methods demonstrated higher rates of detection than parasitological methods, detecting 33 of the 54 (61.1%) known positive samples, while the hematocrit centrifugation technique (best parasitological test) detected only 44.4%. The serological methods, IFAT and ELISA, detected seropositivity in 51 of the 54 (94.4%) and 49 of the 54 (90.7%) known positive samples, respectively. Despite being highly sensitive, the latter only demonstrates exposure to the infectious agent and does not indicate whether the infection is active. The present study was the first to use the qPCR for a South American isolate, improving disease detection and quantification. Furthermore, the analyses revealed that the patent phase of the disease may extend up to 42 days, longer than previously reported. The combination of several diagnostic techniques can lower the frequency of false negative results and contributes toward better disease control.

Follow-up of dairy cattle naturally infected by Trypanosoma vivax after treatment with isometamidium chloride

“…While Kappa results between LAMP and PCR (0.10) and LAMP and ELISA (0.02) indicate slight agreement, it showed a poor agreement (-0.02) between PCR and ELISA. These results agree with those obtained by Fidelis et al (2019), which evaluated different techniques for disease diagnoses, obtaining a Kappa index of 0.07 between PCR and ELISA. The low Kappa values observed in the present study represent a reflection of the different detection rates obtained, which are inherent to different detection targets of each technique.…”

“…Low concentrations of T. vivax DNA related to low parasitemia may also affect the PCR outcomes (Morlais et al, 2001;Cadioli et al, 2015, Fidelis et al, 2019. In the present study, LAMP positive samples presented high mean 6/12 Follow-up after Trypanosoma vivax treatment Cq (39.4), which indicates that positive animals experienced low T. vivax parasitemias after treatment, thus justifying PCR failure.…”

“…In the present study, LAMP positive samples presented high mean 6/12 Follow-up after Trypanosoma vivax treatment Cq (39.4), which indicates that positive animals experienced low T. vivax parasitemias after treatment, thus justifying PCR failure. Studies previously performed by Cadioli et al (2015) and Fidelis et al (2019) showed that in the chronic/sub-patent phase of infection, T. vivax tends to present very low parasitemia, which difficult disease diagnosis in herds.…”

“…Herein, the presence of PCR inhibitors was discarded once all samples tested were positive for the GAPDH gene, thus ensuring the samples' quality and the integrity of the extracted DNA. Fidelis et al (2019) showed that PCR and real-time PCR had the same detection rates for T. vivax; instead, qPCR is useful to determine parasitic load but much more expensive than conventional PCR.…”

“…However, the diagnosis becomes challenging due to the lack of nonspecific clinical signs (Cadioli et al, 2012;Fidelis et al, 2016) and the presence of low parasitemia or aparasitemic periods (Fidelis et al, 2016). Serological methods, such as ELISA, represent an essential tool for screening of T. vivax exposure at the herds level (Cuglovici et al, 2010), but do not allow neither to discriminate animals with active infection from those treated and cured (Nantulya, 1990) nor to identify infected animals during the first days of infection (Fidelis et al, 2019). On the other hand, molecular methods, such as PCR (Cortez et al, 2009;Pimentel et al, 2012) and LAMP (Njiru et al, 2011), show a high capability for T. vivax DNA detection, even in periods supposedly considered as aparasitemic (Cadioli et al, 2015).…”