Molecular detection of Anaplasma marginale in stable flies Stomoxys calcitrans ( Diptera : Muscidae ) feeding on a tick-free bovine herd

Distributed under Creative Commons CC-BY 4.0 Abstract Data on the implication of the stable fly (Stomoxys calcitrans, Diptera: Muscidae), in the transmission of Anaplasma marginale in tick-free cattle is scarce. Hence, the objective of this investigation was to detect the presence of DNA from A. marginale in stable flies caught near a bovine herd, which has been maintained free of ticks for 40 years, and occasionally presents some clinical cases of anaplasmosis. Twenty-four batches of S. calcitrans (15 flies each) were collected in the morning and in the afternoon twice a week (except for one catch in one week of September and one catch in one week of December) during 12 catching days in a period of four months. Their DNA was obtained and analysed by nested PCR (nPCR) to identify the presence of A. marginale DNA. Seven of the batches (29.16 %) were positive for A. marginale, as detected by an nPCR that targets the A. marginale msp5 gene. Similarly, in two out of 12 catching days (16.66 %), those S. calcitrans batches collected in the morning and in the afternoon, were positive for A. marginale, while those S. calcitrans groups collected in three out of 12 catching days (25 %), were positive for the rickettsia. The obtained results suggest that A. marginale transmission is carried out mechanically by S. calcitrans, favouring the circulation and maintenance of the microorganism in this particular bovine herd.


Introduction
2][3] In this context, anaplasmosis, together with bovine babesiosis, causes economic losses higher than 10 billion dollars worldwide. 4 After transmission to bovines, the rickettsia multiplies in their erythrocytes. 4In the acute infection phase, the rickettsemia may reach 10 9 infected erythrocytes (IE)/mL of blood. 5The main clinical signs of anaplasmosis are anaemia, weight loss, and abortion, and death is common. 5Recovered bovines become long-lasting carriers (clinically healthy) in which the rickettsia is very difficult to detect in blood smears examined with the optical microscope. 5,6t is assumed that haematophagous flies mechanically participate in the transmission of A. marginale to the bovines, mainly during the acute phase of the infection in specific areas of the U.S.A. 6 Furthermore, in some areas of Brazil 7 and Costa Rica 8 there is strong evidence that S. calcitrans actively participates in the mechanical transmission of A. marginale to cattle.In this connection, the minimal infectious dose (MID) of A. marginale-infected erythrocytes required for mechanical transmission is unknown.It has been suggested that it is in the range between the minimum of 1 IE and 100 IE. 6 Approximations of the amount of blood transported on stable fly mouthparts range from 0.029 mL to 0.4 mL. 9,10At an MID of 100 IE, rickettsemias between 2.5 X 10 8 (or 10 8.4 ) IE/mL and 3.5 X 10 9 (or 10 9.5 ) IE/mL would be necessary for a single fly to transport 1 MID.In fact, outbreaks of bovine anaplasmosis have been documented in some geographical areas where there are no ticks. 11. calcitrans, is one of the biting flies implicated in the mechanical transmission of A. marginale among bovines, 12 but there are not published reports that demonstrate this assumption.On this basis, the aim of this study was to detect DNA from A. marginale in S. calcitrans caught at 6 meters near a bovine herd, housed in pens with cement floors, which has been maintained free of ticks for 40 years, and occasionally experienced some cases of anaplasmosis.

Sampling site
The study was carried out at an animal facility located at Progreso, Jiutepec, Morelos State, Mexico.It stands at 18º 53´ N, 99º 10´ W, where the average warmest temperatures oscillate between 21.3 ºC (January) and 26.6 ºC (May), while the average coldest temperatures oscillate between 6.5 ºC (January) and 12.5 ºC (June).This facility houses a herd of 80 Aberdeen Angus cattle (65 females, 15 males) whose zootechnical function is meat production.Animals are housed in pens with concrete floors and they are fed with lucerne and a commercial supplement; and water is consumed ad libitum.This herd has sporadically experienced cases of anaplasmosis, and thus the bovines were examined every week to verify they have no ticks.There are a few farmers owning small numbers of cattle and horses in the vicinity.

Sampling of flies
Groups of 15 S. calcitrans were caught without touching them, when the flies were resting after a blood meal on the walls close (6 meters) to the cattle pens.Flies were caught by using a new sterile 50 mL plastic tube, containing 3 mL of sterile distilled water.A tube was slowly and carefully placed on a fly resting on the wall, and then the tube lid was screwed for trapping the fly.Afterwards, the tube was shaken to wet the fly wings and avoid it to scape when the tube was opened again to trap another fly.This procedure was repeated until 15 flies were captured in each tube.
Catches were done in the morning and in the afternoon, twice a week (except for one catch in one week of September and one catch in one week of December) for a total of 12 catching days during September, October, November and December 2015.Flies were identified as Stomoxys calcitrans according to morphological characteristics (seven to eight mm in length and look like house flies). 13They can be distinguished by belly marks in the form of chessboard and by the mouthparts for skin piercing (Figure 1A). 13Only those stable flies showing evidence by visual inspection of having taken a blood meal were collected (Figure 1A -photograph obtained by Carlos R. Bautista-).It is worth mentioning that no other haematophagous dipterans were identified at the time of the study.Immediately after capture, batches of 15 flies were placed in an Eppendorf vial (2 mL), and then the specimens were anesthetized through cold to manipulate them until processed.Whole ground fly batches were stored at -70 °C and prepared as described by Scoles et al. 6 DNA extraction was carried out using the UltraClean™ BloodSpin™ DNA Isolation kit (MO BIO Laboratories Inc., Carlsbad, CA, U.S.A.) following the manufacturer´s procedure.
Samples were tested for A. marginale DNA using a nested PCR (nPCR) that targets the amplification of a fragment from a single copy gene that encodes the outer major surface protein MSP5 from A. marginale.Reaction and amplification conditions were carried out as described by Torioni et al, 13,14 using appropriate positive and negative controls.Sequences for the primers used were based on Torioni et al. 13 and the expected amplicons are described in Table 1.
Amplification conditions in a C1000 Touch™ Thermal cycler (BIO-RAD Laboratories, Inc., CA, U.S.A.) were: 35 cycles 95 °C for 3 minutes, followed by 35 cycles at 95 °C for 30 seconds, 35 cycles at 65 °C for 1 minute, and 35 cycles at 72 °C for 1 minute.A final extension step at 72 °C for 10 minutes was used, which was followed by 35 cycles at 4 ºC, at infinitum.The results of the PCR were analyzed in a 21 % agarose gel stained with Ethidium bromide.Samples were considered positive when amplification of a fragment corresponding to the expected amplicon size was visualized under ultraviolet light (Figure 1B).

Results and discussion
Seven out of 24 S. calcitrans groups were positive for A. marginale as detected by a nested PCR, and an expected amplicon size of 466 bp (Figure 1B).This represented 29.16 % of all the samples (Figure 2A).It was also observed that on two occasions, the S. calcitrans batches collected on the same day (morning and afternoon of October 22 nd and of November 4 th ), were positive for A. marginale (16.66 %), while those S. calcitrans batches collected in the afternoon of October 1 st , morning

Figure 1 .
Figure 1. A. Example of a stable fly Stomoxys calcitrans caught in this study (photograph: Carlos R. Bautista).B. Agarose gel at 2 % stained with Ethidium bromide, which shows the amplification products of msp5 of A. marginale by nested PCR.Lane 1: markers; lane 2, positive control; lanes 3 to 9, Stomoxys calcitrans samples; lane 10, negative control.(Sample in well 6 is positive, although the line is faint).