Six Selected Contributions: Trypanosomiasis and Cryptobiosis

African Trypanosomiasis

Trypanosoma brucei rhodesiense in the blood of a patient, EATRO, Torro, Uganda (1970) Haematocrit Centrifuge Technique, human trypanosomiasis survey, SRI, Tanzania (Woo, 1971) Human trypanosomiasis (gambian sleeping sickness), EATRO, Torro, Uganda (Woo, 1970b)

(1) Woo, P.T.K. 1970a. Origin of mammalian trypanosomes which develop in the anterior station of blood-sucking arthropods. Nature 228: 1059-1062.
He (Woo, 1970a) advanced the hypothesis that pathogenic trypanosomes of mammals in Africa originated recently from leech-transmitted trypanosomes of aquatic reptiles. In a concurrent study, Woo & Soltys (1969) successfully infected reptiles with the pathogenic Trypanosoma brucei and there were no clinical signs associated with the infection - this experimental study supported the hypothesis, and it also suggested that reptiles could be reservoir hosts for human trypanosomiasis in Africa as tsetse flies of the Glossina palpalis group would feed readily on both reptiles and humans. Molyneux (1973) confirmed that African reptiles, especially the Agama lizards could be experimentally infected with parasites from patients with trypanosomiasis. The isolation of pathogenic mammalian trypanosomes from monitor lizards in an endemic area for human trypanosomiasis confirmed that reptiles are reservoir hosts (Njagu et al., 1999). Also, monitor lizards experimentally infected with the trypanosome isolates did not have clinical disease and their trypanosomes were infective to tsetse flies. These studies may have important implications on future strategies to control the disease, and also provide an alternative explanation to some previous outbreaks of human trypanosomiasis in Africa.

(2) Woo, P.T.K. 1970b. The haematocrit centrifuge technique for the diagnosis of African trypanosomiasis. Acta Tropica 27: 384-386.
One of his early contributions on human trypanosomiasis in Africa was to use the Haematocrit Centrifuge Technique (HCT; Woo, 1969) in hospitals to detect trypanosomes in the blood and cerebral spinal fluid of patients with trypanosomiasis (Woo, 1970b). He adapted the HCT for use under field conditions (Woo, 1971) and used it in a survey on human trypanosomiasis (Onyango & Woo, 1971). "The . . . technique of Woo is the most suitable for rural hospitals" (Foulkes, 1981), and it " . . . is still in use in many HAT [Human African Trypanosomiasis] control programs" in Africa (Chappuis et al., 2005). It is also used to diagnose the disease in patients outside of Africa (Lejon et al., 2003), for the detection of congenital Chagas' disease caused by Trypanosoma cruzi in South America ( Moretti et al. 2005), and is ". . . recommended for the parasitological diagnosis of acute Chagas' disease and malaria" in rural areas (Fuente et al., 1985).

Since it is a rapid and sensitive technique (Woo & Rogers, 1974) it is also " . . . widely used for the diagnosis of animal trypanosomiasis" in livestocks (Schlater & Van Den Bossche, 2004), and for detecting trypanosome infections in other animals including fish ( e.g. Bower & Woo, 1979), frogs (e.g. Woo, 1983) and birds (e.g. Wiehhn et al., 1999). The HCT is also a useful tool to determine the effectiveness of chemotherapy against mammalian trypanosomiasis (e.g. Sekoni & Rekwot 2003), and it has been modified to detect pathogenic haemoflagellates (Cryptobia, Spironucleus etc.) in economically important fish (e.g. Woo, 2003 ; Guo & Woo, 2004).

The HCT is sometimes also called the "Woo test" (Chappuis et al., 2005), or "Woo technique" (Quispe et al., 2003) , or "Woo method" (Uilenberg, 1998; Schlater & Van Den Bossche, 2004).
Chappuis F et al. (2005) Options for field diagnosis of human African trypanosomiasis. Clinical Microbiol. Reviews 18: 133-146.
Quispe AP et al. (2003) Prevalencia de Trypanosoma vivax en bovinos de cuatro distritos de la provincia de Coronel Portillo, Ucayali. Rev investig. vet. Peru 14: 161-165.
Uilenberg G (1998) Diagnosis - Chapter 3. In: A field guide for the diagnosis, treatment and prevention of African animal trypanosomosis, Food and Agriculture Organization, UN, 158 pages.
Schlater J & Van Den Bossche P (2004) Trypanosomosis - Chapter 2.3.15. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th edition (eds. Cullen CA & Pearson JE), World Organization for Animal Health, OIE, 1178 pages.

Salmonid Cryptobiosis

Cryptobia (T.) salmositica with a red blood cell from an anaemic fish Bilateral exophthalmia in acute salmonid cryptobiosis (Woo, 2003) General edema and abdominal distension with ascites (Woo, 1979)

(3) Woo, P.T.K. 1979. Trypanoplasma salmositica: experimental infections in rainbow trout, Salmo gairdneri. Experimental Parasitology 47: 36-48.
Woo (1979) described and characterized a disease in salmonids caused by the hemoflagellate, Cryptobia (Trypanoplasma) salmositica, and clinical signs in cryptobiosis include exophthalmia, anemia, anorexia, general edema and abdominal distension with ascites. The pathogen has been reported from all species of Pacific salmon in streams and rivers on the west coast of North America. He has established and maintained an on-going research program (1974 - present) on "Cryptobia and cryptobiosis" and has published extensively on it. The three main objectives of the program (Woo, 2003) are to (1) better understand the biology of the pathogen, (2) elucidate the host-parasite relationships which include the disease mechanism, and (3) develop rational strategies against the pathogen and disease. Protective strategies have been developed ( Woo, 2010) and they include exploiting (a) innate (natural) immunity [breeding of Cryptobia-resistant fish (Forward et al., 1995)], (b) adaptive (acquired) immunity [development of a live vaccine (Woo & Li, 1990) and a DNA vaccine (Tan et al. 2008)], and (c) therapy [chemotherapy (Ardelli and Woo 2001a; Ardelli and Woo 2001b) and immunochemotherapy (Ardelli and Woo 2001c)] against the parasite. This 'proof-of-concept' research program is also used to train numerous highly qualified personnels (postdoctoral fellows, graduate students, technicians and undergraduate students). Other collaborators include colleagues and visiting scientists - they worked on cryptobiosis partly because of the interdisciplinary nature of the program.

(4) Woo, P.T.K. & Li, S. 1990. In vitro attenuation of Cryptobia salmositica and its use as a live vaccine against cryptobiosis in Oncorhynchus mykiss. Journal of Parasitology 76: 752-755.
This is the first vaccine against a fish parasite - the live vaccine injected intraperitoneally into fish circulates in the blood of rainbow trout for at least 6 months, does not cause disease but protects fish from cryptobiosis (Woo & Li, 1990). A single dose of the vaccine protects rainbow trout (adaptive immunity) for at least 2 years, and protection is through the production of complement fixing antibodies, enhanced phagocytic activities by marcophages, and cell-mediated cytotoxicity (both antibody-independent and antibody-dependent) (Li & Woo, 1995). Since this vaccine has no detectable bioenergetic costs to juvenile fish and does not affect growth (Beamish et al., 1996) it is used routinely to study the acquisition and mechanism of protective immunity in salmonids (Woo, 2001). The vaccine has remained attenuated (does not secrete metalloprotease, the main virulent factor in cryptobiosis) and it protects both juvenile and adult salmonids from cryptobiosis (Woo, 2003). The two strains (vaccine and pathogen) have been used to detect Atlantic salmon that have a more responsive immune system (Chin et al., 2004) - aims of the study are to identify genes in salmon related to a rapid response to pathogens, and to emphasize this trait in breeding programs. This is a proactive strategy against pathogenic organisms.

(5) Forward, G.M., Ferguson, M.M. & Woo, P.T.K. 1995. Susceptibility of brook charr, Salvelinus fontinalis to the pathogenic haemoflagellate, Cryptobia salmositica, and the inheritance of innate resistance by progenies of resistant fish. Parasitology 111:337-345.
Using a very host specific hemoflagellate (Cryptobia catostomi) from white suckers, Bower & Woo (1977) showed the Alternative Pathway of Complement Activation (APCA) operates in the blood of fish. They described an in vitro technique to determine in vivo susceptibility of fish to parasites. APCA is innate or natural immunity and it also operates in other teleosts; for example, against Cryptobia salmositica ( Wehnert & Woo, 1980), a pathogenic hemoflagellate in salmonids. In salmonid cryptobiosis (Woo 1979) some brook charrs are innately resistant to C. salmositica, and this resistance to infection is inherited and controlled by a dominant Mendelian locus. Cryptobia-susceptible charrs are homozygous recessive while Cryptoba-resistant fish are either homozygous dominant or heterzygous; hence it is now possible to breed C. salmositica-resistant charrs (Forward et al., 1995) where the parasite is ruptured via APCA (Forward & Woo, 1996). The adaptive or acquired immune (both humoral and cell-mediated) responses to various antigenic stimulations (including to a commercial Aeromonas vaccine) are very similar in both Cryptobia-resistant and Cryptobia-susceptible charrs (Ardelli & Woo 1995). APCA is an important protective mechanism in fish against pathogenic protozoa (Woo 2007) and attempts are being made to exploit this form of innate immunity.

(6) Tan, C.W., Jesudhasan, P. & Woo, P.T.K. 2008. Towards a metalloprotease-DNA vaccine against piscine cryptobiosis caused by Cryptobia salmositica. Parasitology Research 102: 265-275.
The pathogenic strain of Cryptobia salmositica has a cysteine protease and a metalloprotease (Zuo & Woo, 1997a). The cysteine protease (49, 60, 66 and 97 kDa) is a metabolic enzyme while the metalloprotease (200 kDa) is the disease-causing factor in cryptobiosis (Zuo & Woo, 1997a). The purified metalloprotease digests red call membrane, collagens and laminin (Zuo & Woo, 1997b; Zuo & Woo, 2000). A murine monoclonal antibody (mAb 001; Feng & Woo, 1996) against a 200 kDa glycoprotein epitope (Feng & Woo, 1998a; 1998b) is protective (both prophylactic and therapeutic) when injected into fish (Feng and Woo, 1997). MAb001 does not fix complement to lyse the pathogen but agglutinates it (Feng & Woo, 1996), inhibits both parasite multiplication and respiration (Hontzeas et al., 2001), and neutralizes the activities of the metalloprotease (Zuo et al. 1997). The cysteine (Jesudhasan et al., 2007a) and metalloprotease (Jesudhasan et al., 2007b) genes have been sequenced and DNA vaccines produced by inserting the protease genes into plasmids (Tan et al., 2008). The metalloprotease-DNA (MP-DNA) vaccine (secretes metalloprotease) after inoculation into fish (Oncorhynchus mykiss and Salmo salar) caused an anemia but the PCVs of fish started to recover as antibodies were produced to neutralize the secreted metalloprotease. The antibodies also agglutinated live Cryptobia; however, fish inoculated with cysteine-DNA plasmids (or control fish with plasmids alone) were not anemic and they did not have agglutinating antibodies against Cryptobia. As expected MP-DNA-vaccinated fish were not protected from infection after parasite challenge, but they had lower parasitemias, delayed peak parasitemias, and faster recovery compared to control fish. However, fish vaccinated with attenuated strain (Woo & Li, 1990) were protected and this confirmed the two vaccines (attenuated and the metalloprotease DNA) operated through different mechanisms. In a review on the use of DNA vaccines in aquatic organisms Kurath (2008) confirms this is the ". . . first published demonstration of protective effects of a fish parasite DNA vaccine in fish".