Real time polymerase chain reaction to diagnose Anaplasma marginale in cattle and deer (Ozotoceros bezoarticus leucogaster) of the Brazilian Pantanal

Epizootiological study of Anaplasma marginale in regions that contain various reservoir hosts, co-existence of rickettsia pathogens, and common vectors is a complicated task. To achieve diagnosis of this rickettsia in cattle and campeiro deer of Brazilian Pantanal, a comparison was made between a real time polymerase chain reaction (RT-PCR) with intercalating Sybr Green fluorochrome and primers based on msp5 gene of A. marginale; a conventional PCR (C-PCR); and parasitological examination using thin blood smear stained with Giemsa-MayGrunwald. Both PCRs showed good performance in the diagnosis of A. marginale in cattle, and were superior to the parasitological exam. The RT-PCR detected seven positive campeiro deer (16.3%). This rate was significantly higher compared to C-PCR, which identified one animal as positive (2.3%), and also compared to parasitological diagnosis, which did not find any positive animals. The dissociation temperature average of positive reactions in cattle (81.72 °C ± 0.20) was identical to dissociation temperature found in the cervids (81.72 °C ± 0.12), suggesting that both animal species were infected with A. marginale. We concluded that RT-PCR can be used for A. marginale diagnosis and in epizootiological studies of cattle and cervids; in spite of the small number of campeiro deer samples, the results indicated that this wildlife species has importance in the Anaplasma epizootiology in the Brazilian Pantanal.

The intraerythrocytic Anaplasma sp microorganisms of the Rickettsiales order and Anaplasmataceae family (DUMLER et al., 2001) cause in susceptible animals fever, anemia, icteric, anorexia, weight loss, and abortion (KOCAN et al., 2003). Anaplasma marginale produces considerable economic losses to the cattle industry of tropical and subtropical regions of the world due to significant productivity reduction and mortality (KOCAN et al., 2003). In regions where wild ruminant populations are abundant, particularly cervids, which have been implicated as important reservoirs and infection sources of A. marginale (DE LA FUENTE et al., 2008), epizootiological studies of this rickettsia are more complicated due to various reservoir hosts, co-existence of rickettsia pathogens, and common vectors. As well as being infected by A. marginale, the cervids are also infected with Anaplasma phagocitophylum (DUMLER et al., 2001) and Anaplasma ovis (HAIGH et al., 2008), which makes it difficult to implement a surveillance by serology. The current serological tests display crossed reactions, even when antigenic sub-units such as MSP5 recombinant protein are employed as antigens, because Anaplasma species have similar B epitopes (STRIK et al., 2007).
Several molecular diagnostic methods with high levels of sensitivity and specificity are used, such as reverse line blotting (GUBBELS et al., 1999), conventional polymerase chain reaction (C-PCR) (FRENCH et al., 1998), semi-nested, nested PCR (MOLAD et al., 2006) and real time PCR (RT-PCR) (DECARO et al., 2008). This last technique presents some advantages, such as speed of obtaining results, quantification of the infection level, and a minor probability of contamination. So far, the RT-PCR that has been developed to detect anaplasma in bovines uses the Taqman system as a reaction indicator (DECARO et al., 2008). This system requires a specific probe with a fluorescent reagent and a fluorescence emission quencher, making it more expensive than the technique that utilizes double strand DNA intercalating fluophore, the Syber Green. Because previously RT-PCR with primers based on genes msp5 and msp2 and Syber Green were used only to detect A. marginale gene transcription in the tick vector (FUTSE et al., 2003), this work evaluated a RT-PCR using primers with sequences of the msp5 gene with Sybr Green for diagnostic and epizootiological study of A. marginale in bovines and cervids.
To assess RT-PCR in the diagnosis of A. marginale in bovines, three experimental groups were established according to the following descriptions.Group 1 consisted of ten Brangus calves raised in the isolation area at the Embrapa Gado de Corte, Campo Grande, Mato Grosso do Sul State, seven to nine months of age, serologically negative in enzyme linked immunosorbent assay with recombinant proteins of A. marginale, MSP2 (ARAÚJO et al., 2005), and MSP5 (SILVA et al., 2006) and in conventional PCR (C-PCR) (ARAÚJO et al., 2005). Group 2 consisted of ten Brangus calves raised in an endemic area for A. marginale, ratified as positive by the above diagnostic tests. Group 3 consisted of eight Brangus calves of the same age and raised in the same isolation area, confirmed as negative through the above diagnostic tests and immunized with attenuated Babesia bovis and B. bigemina to avoid babesiosis sintomatology. These calves subsequently were exposed to A. marginale infection by ticks (20 engorged PIOVEZAN et al. (2006). The A. marginale direct diagnosis was performed by thin blood smears from capillaries of calves' ears and stained with May-Grunwald Giemsa, C-PCR according to ARAÚJO et al. (2005), and by RT-PCR. For the RT-PCR procedure, the reaction was obtained in a 25 µL volume containing PlatinunSYBR Green qPCR SuperMix-UDG (Invitrogen), primers forward 5'-AAGGCGAGGAGCTGTTTAAG-3' and reverse 5'-CTACTGCCTCACAAGGACGA-3' based on the sequence of the gene msp5 of A. marginale, at concentration of 20 ηg.µL -1 , and DNA at concentration of 5 ηg.µL -1 . Three stages were programmed in the thermocycler (Cepheid Smart Cycler): 1) one cycle at 95 °C for 600 seconds; 2) 35 cycles at 95 °C for 15 seconds and 52 °C for 45 seconds; and 3) 60 cycles at 95 °C, for two hundredths of a second, to create the dissociation curve or melting curve. The statistical analysis was done by McNemar test.
The RT-PCR standardization with DNA samples obtained from blood of A. marginale infected cattle, showed positive reactions, while blood samples from animals free of this rickettsia infection did not show amplicons. The same results were verified in the C-PCR. In the calves of group 3, RT-PCR and C-PCR showed similar results, with the first A. marginale detection by both PCRs at Day 21 after exposure of the calves to tick or insect transmission, while in the peripheral blood smears A. marginale was observed at the 26 th day post challenge. The only disagreement between the PCRs occurred with the sample from calf number 6, at 59 days post challenge, which displayed positive reaction only in the C-PCR.
The campeiro deer thin blood smear examination did not detect A. marginale. Using C-PCR one animal was detected as positive (2.3%), while RT-PCR found seven positive reactions (16.3%), which resulted in a significant statistical difference between the molecular diagnostics. Therefore, RT-PCR using Sybr Green demonstrated superior performance as a diagnostic tool.
The RT-PCR described here has advantages over the recently developed RT-PCRs (DECARO et al., 2008), since they employ a Taqman system, which increases the tests costs, compared to method that uses the intercalating fluorochrome of DNA double strand. Another advantage is the use of the dissociation temperature, which confers a diagnostic specificity that is important for epizootiological studies.
There is serological evidence that cattle infection with A. phagocitophylum occurs in areas endemic for A. marginale (DE LA FUENTE et al., 2008), as well as deer with infections of A. phagocitophylum and A. ovis (DUGAN et al., 2006). However, crossed reactions in serological tests (even when using recombinant protein MSP5;STRIK et al., 2007), due to common antigens between the species of Anaplasma (DREHER et al., 2005), do not allow a conclusive diagnosis of Anaplasma species infecting cattle and cervids. Also, the gene msp5 is considered highly conserved between Anaplasma species (KNOWLES et al., 1996), consequently there is possibility of amplicon production in the PCR with primers based on this gene sequence for different Anaplasma species.
However, msp5 gene blast of different Anaplasma species displays different degrees of identity in the gene sequence; for instance Picoloto, G. et al. Rev. Bras. Parasitol. Vet. A. marginale msp5 has 89, 85, and 65% of identity, respectively, with ortologous genes of A. ovis, A. centrale, and A. phagocitophylum. It is likely that the RT-PCR melting curve would show distinct temperature dissociation for each Anaplasma species. Since the average temperature of dissociation in positive reactions in cattle (81.72 ± 0.20) (Figure 1) and in campeiro deer (81.72 ± 0.12) were identical, there is the suggestion that both animal species were infected with A. marginale. This finding agrees with the fact that only A. marginale was found infecting deer in Brazil by a direct diagnosis with PCR (MACHADO et al., 2006).
In conclusion, this RT-PCR showed good performance as a diagnostic test for A. marginale in cattle and in campeiro deer, as suggested by dissociation temperature. Despite the small number of campeiro cervid samples, the results pointed to the importance of these animals as Anaplasma reservoirs in the Brazilian Pantanal. A complementary study on dissociation temperature of A. ovis, A. centrale, and A. phagocitophylum in this RT-PCR would be relevant for its use in epizootilogical studies of Anaplasma sp., in regions or farms where various animals species co-exist.