Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method

Abstract

Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with 13 considerable worldwide economic, medical, and veterinary impact. Babesiosis and other 14 TBDs were considered responsible for 50% of the deaths of cattle occurred in 15Mozambique in the first year after importation from neighbouring countries. 16Here, we present the detection of B. bigemina and B. bovis in cattle from Mozambique 17 and the development of a novel PCR method. For this study, blood samples were 18 collected in one farm located near Maputo city. The samples were analyzed using a 19 previously described nested PCR and a novel hot-start PCR method. Primers were 20 selected for the hot-start PCR based on the putative gene of an undescribed aspartic 21 protease named babesipsin, present in both B. bovis and B. bigemina. The combination of 22 hot start polymerase and long primers (29-31bp) were in this study determinant for the 23 Manuscript 2 successful amplification and detection in only one PCR. The babesipsin hot-start PCR is 24 a fast, simple, and sensitive method, with some advantages over current methods. A total 25of 117 field samples were tested by hot-start PCR, and 90 were positive for B. bigemina 26 (77%), 82 were positive for B. bovis (70%), 61 were mixed infections (52%) and only 6 27 were negative (5.1%). The results confirm that this area of Mozambique is endemic for 28 Babesiosis, and that this TBD should be regarded as a threat for imported cattle. 29 30

Risk factors associated with the frequency of antibodies against Babesia bovis and Babesia bigemina in cattle in southern Mozambique

“…In regions in eastern and southern Africa, studies indicate that bovine babesiosis presents seropositivity ranging from 19.5 to 94.0% of the cattle (Dreyer et al 1998, Mbata et al 2002, Mtshali et al 2004, Martins et al 2008. In South Africa, these studies showed that the prevalence of antibodies against Babesia bigemina ranged from 62.4 to 94.0%, and in the case of B. bovis the frequency was 19.5%.…”

“…Na província de Gaza a soropositividade foi de 67,5% (280/415) para B. In the mid-1990s the government of Mozambique implemented a livestock restocking program based on the import of cattle from the Republic of South Africa and Zimbabwe (Martins et al 2008). However, this increase in the production of ruminants in the country was not accompanied by improvements in sanitary conditions, what led to the occurrence of different diseases including the following tick-borne conditions: Babesiosis, anaplasmosis, ehrlichiosis and theileriosis.…”

“…Thus, infections with Babesia spp. in cattle are characterized by fever, anemia, hemoglobinemia, hemoglobinuria, and in many cases, death (Martins et al 2008).…”

Diagnostic approaches for tick-borne haemoparasitic diseases in livestock

“…As mentioned earlier, RLB technique has proved highly sensitive and specific for the detection of these parasites (Gubbels et al, 1999). A hot-start PCR (semi-nested) to detect B. bovis, B. bigemina based on aspartic proteinase babesipsin gene was developed and successfully applied (Martin et al, 2008;Awad et al, 2011). Multiplex PCR (Figueroa et al, 1993), real-time PCR (Buling et al, 2007) and multiplex LAMP (Iseki et al, 2007) are in process of development and validation for identification and characterization of B. bigemina.…”

Loop-mediated isothermal amplification (LAMP) detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758) in China

“…et al, 1993;Smeenk et al, 2000;Almeria et al, 2001;Oliveira-Sequeira et al, 2005;Liu et al, 2007;Martins et al, 2008;Sibeko et al, 2008). These techniques, however, rely on the availability of expensive equipment such as thermocyclers.…”