**2. Field isolation of trypanosomes**

Trypanosomes are isolated from infected hosts during active or passive disease surveillance activities. The infected hosts include humans, domestic and wild animals, as well as tsetse fly vectors. Parasites are isolated from biological fluids including blood, cerebrospinal fluid (CSF) and lymph node aspirates, and/or body parts of tsetse fly vectors. Depending on the host parasitemia and/or density of trypanosomes in the biological fluids at the time of isolation, trypanosomes can be either cryopreserved directly or propagated in immunosuppressed laboratory rodents prior to cryopreservation.

#### **2.1. Diagnosis: buffy coat and whole blood parasitemia**

Parasitological diagnosis of trypanosome infections in animals and humans can be made through microscopic examination of wet blood smears, stained thin and thick blood smears, smears of lymph node aspirates, and buffy coats [11]. Under normal field conditions when large numbers of animals are sampled, examination of buffy coats, obtained through capillary tube centrifugation technique (CTC) [12], is the preferred method of diagnosis due to its higher sensitivity compared to other microscopic techniques. Animals suspected to be infected with trypanosomes are bled from the ear vein into heparinized capillary tubes after which the 3/4 full capillary tubes are sealed at one end with plasticine and then spun in a hematocrit centrifuge at 10,000 revolutions per minute for 5 min. Blood separates into three portions, namely, the red blood cells, which settle at the bottom of the capillary, the plasma portion found at the top, and the buffy coat portion, which forms at the interface of the red blood cells and plasma. Trypanosomes are concentrated in the buffy coat portion of the centrifuged blood, thus enhancing the sensitivity of the test. In humans, diagnostic methods that are routinely employed to detect blood trypanosomes include (CTC), quantitative buffy coat (QBC), mini anion exchange centrifugation technique (mAECT), and modified mAECT [13]. For the diagnosis of trypanosomes in cerebrospinal fluid, available methods include single and double centrifugation and modified single centrifugation (MSC), with the MSC being easy to perform and as sensitive as the double centrifugation [13].

Once confirmed positive, the density of trypanosomes in the relevant biological, fluid is determined. Whole blood is drawn from the jugular vein of the infected host into anticoagulant containing tubes and used to quantify the parasitemia using the matching method [14] for the Trypanozoon group of trypanosomes. Direct isolation is therefore determined by whole blood parasitaemia.

#### **2.2. Direct isolation of parasites from infected biological fluids**

Parasitemia is usually low in naturally infected hosts. However, the required density of between 3.2 × 107 trypanosomes/ml and 1.3 × 108 trypanosomes/ml may be obtained in a small proportion of the infected hosts, thus permitting direct cryopreservation of the stabilates. In such cases, the infected whole blood is mixed with either of the following cryoprotectants and processed:


Samples are then labeled and dipped into a vapor shipper liquid nitrogen cylinder for transportation from the collection site to the main laboratory for further processing. A vapor shipper is a liquid nitrogen cylinder which has a mechanism of absorbing liquid nitrogen into its system leaving the hollow space full of liquid nitrogen vapor, with temperature in the range of –60 to –80°C. In the laboratory, samples are removed from the vapor shipper and allowed to thaw on ice or at 4°C after which they are dispensed into plain capillary tubes (**Figure 2**), which are then sealed at one end using plasticine.

Isolation and Cryopreservation of Trypanosomes and their Vectors for Research and Development in Resource... http://dx.doi.org/10.5772/65283 7

**Figure 2.** Loading plain capillary tubes with cryo‐protected infected blood sample.

**Figure 3.** Aluminum cane for holding the ampoules.

**2.1. Diagnosis: buffy coat and whole blood parasitemia**

6 Cryopreservation in Eukaryotes

and as sensitive as the double centrifugation [13].

which are then sealed at one end using plasticine.

**2.2. Direct isolation of parasites from infected biological fluids**

**1.** 20% glycerol in EDTA saline glucose (ESG), pH 8.0 in the ratio of 1:1.

parasitaemia.

processed:

Parasitological diagnosis of trypanosome infections in animals and humans can be made through microscopic examination of wet blood smears, stained thin and thick blood smears, smears of lymph node aspirates, and buffy coats [11]. Under normal field conditions when large numbers of animals are sampled, examination of buffy coats, obtained through capillary tube centrifugation technique (CTC) [12], is the preferred method of diagnosis due to its higher sensitivity compared to other microscopic techniques. Animals suspected to be infected with trypanosomes are bled from the ear vein into heparinized capillary tubes after which the 3/4 full capillary tubes are sealed at one end with plasticine and then spun in a hematocrit centrifuge at 10,000 revolutions per minute for 5 min. Blood separates into three portions, namely, the red blood cells, which settle at the bottom of the capillary, the plasma portion found at the top, and the buffy coat portion, which forms at the interface of the red blood cells and plasma. Trypanosomes are concentrated in the buffy coat portion of the centrifuged blood, thus enhancing the sensitivity of the test. In humans, diagnostic methods that are routinely employed to detect blood trypanosomes include (CTC), quantitative buffy coat (QBC), mini anion exchange centrifugation technique (mAECT), and modified mAECT [13]. For the diagnosis of trypanosomes in cerebrospinal fluid, available methods include single and double centrifugation and modified single centrifugation (MSC), with the MSC being easy to perform

Once confirmed positive, the density of trypanosomes in the relevant biological, fluid is determined. Whole blood is drawn from the jugular vein of the infected host into anticoagulant containing tubes and used to quantify the parasitemia using the matching method [14] for the Trypanozoon group of trypanosomes. Direct isolation is therefore determined by whole blood

Parasitemia is usually low in naturally infected hosts. However, the required density of between 3.2 × 107 trypanosomes/ml and 1.3 × 108 trypanosomes/ml may be obtained in a small proportion of the infected hosts, thus permitting direct cryopreservation of the stabilates. In such cases, the infected whole blood is mixed with either of the following cryoprotectants and

**2.** Glycerol in the ratio of 1:4, that is, one part of the infected blood to four parts of glycerol. Samples are then labeled and dipped into a vapor shipper liquid nitrogen cylinder for transportation from the collection site to the main laboratory for further processing. A vapor shipper is a liquid nitrogen cylinder which has a mechanism of absorbing liquid nitrogen into its system leaving the hollow space full of liquid nitrogen vapor, with temperature in the range of –60 to –80°C. In the laboratory, samples are removed from the vapor shipper and allowed to thaw on ice or at 4°C after which they are dispensed into plain capillary tubes (**Figure 2**),

The loaded and sealed capillary tubes, approximately 18 in number, are then accommodated in a perforated 4.5 ml ampoule tube into which a label is inserted. The label has the laboratory sample identification number. The ampoule normally has two perforations, one at the top and the other at the bottom to allow direct contact of the sample with liquid nitrogen and at the same time continuous flow of liquid nitrogen from the top through the bottom perforations. Before permanent storage, a capillary is removed from the suspended sample and the viability of the trypanosomes confirmed. For ease of permanent storage in liquid nitrogen at –196°C, the ampoule is then placed on to an aluminum cane which has the capacity of holding at least two ampoules: one at the top and the other at the bottom of the cane. The cane is then placed into a canister, with each canister having a capacity of holding 14 canes. The canister is then immersed into liquid nitrogen in the permanent storage dewar (**Figures 3** and **4**) at –196°C.

**Figure 4.** Stabilate storage dewars.

#### **2.3. Isolation and propagation of low parasitemia blood samples in laboratory rodents**

When the parasitaemia in the whole blood of the naturally infected host is low i.e. below 1.3×108 trypanosomes/ml, the anticoagulated infected blood sample is intraperitoneally inoculated into an immunosuppressed laboratory rodent. It is advisable to inoculate two rodents, usually mice, per positive blood sample (isolate). This is carried out in the field where the animals are given identification numbers and transported to the laboratory for monitoring. In the laboratory, the infected mice are maintained on commercial mice pellets (Unga Feeds Ltd., Kenya), provided with water *ad libitum* and monitored for development of parasitemia. At the first peak of parasitemia between 1.3 × 108 trypanosomes/ml and 2.5 × 108 trypanosomes/ ml, the infected mice are euthanized and the blood harvested by cardiac puncture into tubes containing EDTA as anticoagulant. The blood is mixed gently before addition of a cryopre‐ servative at a ratio of 1:1. The samples are suspended in liquid nitrogen vapor for at least 2 h using a cooling jacket before permanent storage in liquid nitrogen at –196°C [15, 16]. Samples with a trypanosome concentration below 6.3 × 107 /ml may also be preserved by direct addition of glycerol to infected biological fluids to a final concentration of 20%, especially if the isolated trypanosome species does not infect rodents [17].
