Isolation of Balamuthia mandrillaris (Free Living Amoeba) from Shatt Al-Arab River in Basrah, South of Iraq

Balamuthia mandrillaris is a free living, opportunistic amoeba was first discovered in 1986 in a mandrill baboon at the Wild Animal Park in California suffered from a neurological disease, later it was associated with many human CNS fatal infection and skin infection all over the world, and it considered to be ubiquitous. We investigate the presence of B. mandrillaris in Shatt Al-Arab, the main river in Basrah south of Iraq, the amoeba was identified morphologically and genetically by PCR. Trophozoite and cyst were observed in culture, the trophozoite with finger like pseudopodia that subdivided into small arms. Rounded cyst of about 13-30 μm surrounded by outer thin wrinkled layer gave the shape of a rose flower. Our finding was the first in Iraq, Balamuthia mandrillaris represent a health hazard in such main river in Basrah.


Introduction
Balamuthia mandrillaris is an opportunistic freeliving amoebae that causes serious cutaneous infections and fatal encephalitis in human (Siddiqui & Khan, 2015) and may invade the skin causing extensive skin lesions (Martinez and Visvesvara, 1997). B. mandrillaris infection has been identified in other mammalian species, such as sheep, horses and dogs (Schuster et al., 2009).
B. mandrillaris was first isolated in 1986, from the brain tissue of a mandrill baboon (Papio sphinx) at the San Diego Zoo Wild Animal Park in California that died after a neurological disease (Visvesvara et al., 1990). Later it was associated with fatal human infections involving the CNS (Anzil et al., 1991). The organism was suggested to enter the body through wounds in the skin that contaminated by soil or by inhalation of cysts carried by wind from soil to the lower respiratory tract (Martinez & Visvesvara, 1997).
The trophozoite is pleomorphic, uninucleated and binucleated forms are occasionally seen (Lokhande et al., 2015). It characterized by the irregular branching pattern (Visvesvara et al., 1993). Balamuthia trophozoite is similar to Acanthamoeba but larger with a specific morphology (Booton et al., 2003b). Balamuthia cyst possessing three walls that seems to be proteinaceous containing mostly cysteine-rich proteins, no polysaccharides or carbohydrate moieties were detected in the cyst wall; mesocyst seems to contain cellular debris such as lipid granules (Klieščiková, 2013). (Schuster, 2002), but thought to be ubiquitous in the environment (MMWR, 2010). Balamuthia and Acanthamoeba supposed to occupy the same ecological habitats and found naturally in soil (Booton et al., 2003b).The first environmental isolation of this amoeba was from soil by Schuster et al. (2003), and recently was isolated from aquatic environment by Lokhande et al., (2015), it was also recorded from infection of two dogs who swam in pond water previously (Finnine et al., 2007).

Material and Methods:
Water samples were collected in 100 ml sterile cups from Shatt Al-Arab River. The date and site details were fixed for each sample. Within the next 24 hours of collection, 2-3 ml of each water sample was cultured on non-nutrient agar medium (2%) in petri-dishes; three replicates were done; incubated at 25Cº for three days at least before examination under light microscope.
Purification was done by using a corck-borer like device, designed by Dr. Muslim A. Rahman, that allow to cut a piece of wet agar medium, of about 0.5 mm diameter under light microscope, Fig.(1), to ensure the presence of targeting amoeba and minimizes the chance for getting other amoeba spp. Then the piece was transported to a fresh wet NNagar medium usually, incubated at 25 Cº for 3 days at least before growth can be observed. This technique was repeated till obtained a pure isolates. The identity of amoeba was confirmed , after morphological characterization, genetically by conventional PCR using a set of B. mandrillaris specific two primers designed by Booton et al., (2003a): 5Balspec16S (5-CGCATGTATGAAGAAGACCA-3) and 3Balspec16S (5-TTACCTATATAATTGTCGATACCA-3) (manufactured by Alpha DNA). DNA was extracted by using chelex resin according to Iovieno et al. (2011), lysis buffer preparation according to Mirhendi et al. (2006). The PCR product yield was a 1,075 bp from mitochondrial small-subunit-rRNA genes in B. mandrillaris according to the following protocol: 40 cycles of 1 min at 94°C, 2 min at 48°C, and 3 min at 72°C followed by a 15min final extension at 72°C . PCR product was electrophoresed on 1.5% agarose gel and visualized by UV.

Results
The trophozoite of B. mandrillaris measuring 30-65 µm, it has a finger-like projections, transparent cytoplasm and a single large nucleus, it moved forward very fast on agar surface by the finger like pseudopodia, the most important identification feature of the trophozoites is the subdivided pseudopodia into small arms, B. mandrillaris trophozoite showed an ability to produce pseudopodia from any part of the amoebic body, Fig. (2, 3 and 4). It is difficult to diagnose B. mandrillaris trophozoites on slide for the first time, because of narrow surrounded space under cover slips, they took the ordinary ovoid appearance of amoeba then the amoebic body elongated and pseudopodium extended and short finger projections appeared, the pseudopodia continued in elongation and sub-branching while the body mass decreased.   Cyst of B. mandrillaris, Fig. (5), was more easy to observed in cultures and on slide, it was rounded of about 13-30 µm surrounded by inner thick regular rounded membrane and outer thin wrinkled one gave the shape of a rose flower, only the endocyst and ectocyst could be recognized under light microscope. The endocyst is rounded without arms and had no pores like Acanthamoeba. Some cysts had rounded smooth outer membrane that may cause a confusing during morphological identification, Fig. (6). The cysts were usually dark brown, which gave the surface of culture media a light brown color like dust. Conventional polymerase chain reaction (PCR) using the species specific primer Balspec16S forward and reverse that amplified a portion of mitochondrial rRNA gene yield a 1057 bp product was done to confirm morphological diagnosis, Fig.(7).

Discussion
Balamuthia mandrillaris is thought to be ubiquitous in the environment (MMWR, 2010). It was supposed to occupy the same ecological habitats of Acanthamoeba and found naturally in soil (Booton et al., 2003b). Acanthamoeba spp. were also recovered from many different ecologies all over the world, it found in any moist environment rich in bacterial source including: brackish, fresh water and tap water (Visvesvara et al., 2007 a and b). Yousuf et al., (2013) isolated Acanthamoeba and Naegleria fowleri from domestic water and recently B. mandrillaris was also isolated from aquatic environment by Lokhande et al., (2015).
The present study is the first in Iraq that detect and isolate this opportunistic free living amoeba from the environment, the primary isolation and purification was depended on cyst mainly, in spite of the large trophozoites size of B. mandrillaris, it was hard to observe and identify on agar or slide, the trophozoite is transparent and had a polymorphosim but the irregular branching and subdivided pseudopodia with distinct cyst morphology are considered helpful features in morphological recognition between B. mandrillaris and some species of Acanthamoeba.
First isolation of B. mandrillaris from environment was from plants pots , where organic fertilizer usually used, this may refer to a need for organic resources or could be in associated with the abundant of bacteria and other organisms in such environment that Balamuthia could feed on. The isolation of B. mandrillaris from river water in this study may reveal pollution with organic residues that encouraged their growth, the water sample was poured directly into NN agar medium without filtration or concentration this reflect the high abundance of B. mandrillaris in river water. Morphological identification was confirm by PCR used genus specific primers designed by Booton et al. (2003a), that do not amplify DNA from other free living amoebas even the closest Acanthamoeba spp.
B. mandrillaris may serve as a biological host as well as a transmission vector for some pathogenic bacteria (A. Matin et al., 2008). As Shatt Al-Arab river is the main source for domestic water and other usage in Basrah, the presence of B. mandrillaris can be considered a health hazard, as B. mandrillaris is an opportunistic amoeba and also may harbor some pathogenic bacteria.