Establishing, Expanding, and Certifying a Closed Colony of Phlebotomus argentipes (Diptera: Psychodidae) for Xenodiagnostic Studies at the Kala Azar Medical Research Center, Muzaffarpur, Bihar, India

Abstract

This pilot project was preliminary and essential to a larger effort to define the ability of certain human-subject groups across the infection spectrum to serve as reservoirs of Leishmania donovani infection to sand flies in areas of anthroponotic transmission such as in Bihar state, India. This is possible only via xenodiagnosis of well-defined subject groups using live vector sand flies. The objective was to establish at the Kala Azar Medical Research Center (KAMRC), Muzaffarpur, Bihar, India, a self-sustaining colony of Phlebotomus argentipes (Annandale & Brunneti), closed to infusion with wild-caught material and certified safe for human xenodiagnosis. Prior to this endeavor, no laboratory colony of this vector existed in India meeting the stringent biosafety requirements of this human-use study. From March through mid-December, 2015, over 68,000 sand flies were collected in human dwellings and cattle sheds using CDC-type light traps over 254 nights. Blood-fed and gravid P. argentipes females were selected and placed individually in isoline-rearing vials for oviposition, and >2,500 egg clutches were harvested. Progeny were reared according to standard methods, providing a continuous critical mass of F1 males and females to stimulate social feeding behavior. With construction of a large feeding cage and use of a custom-made rabbit restrainer, the desired level of blood-feeding on restrained rabbits was achieved to make the colony self-sustaining and expand it to working level. Once self-sustaining, the colony was closed to infusion with wild-caught material and certified free of specific human pathogens.

Keywords: Phlebotomus argentipes; colonization; sand fly; xenodiagnosis.

Fig. 1

Sand fly insectary facility, KAMRC, Muzaffarpur, Bihar, India: (a) colony room showing incubators and environmental cabinets; (b) preparation and processing room; (c) larva food preparation and autoclave room; (d) animal housing.

Fig. 2

Collecting sand flies and keeping them alive: (a) CDC-type light trap installed in a corner of a cattle shed; (b) double-ring fine-mesh collection nets expanded with plastic struts to prevent the nets from collapsing and crushing or injuring the sand flies.

Fig. 3

Initiating the colony through isoline rearing.

Fig. 4

Isoline rearing: (a) Female sand flies were captured into 10–15-ml isoline-rearing vials for egg laying; (b) isoline rearing vials were provided with a small piece of cotton soaked in 30% sucrose solution placed on the screen top of each vial as an energy source and the vials were stored in plastic boxes at 28 °C and 80% humidity.

Fig. 5

Establishing and expanding the colony.

Fig. 6

Custom-made feeding cup used for xenodiagnostic feeds (Precision Plastics, Beltsville, MD).

Fig. 7

Graph showing seasonal profile of sand fly collections using CDC light traps, 1 March to mid-December, 2015.

Fig. 8

Image showing amplified PCR products derived from DNA of colony sand flies on agarose gel, confirming their identity as P. argentipes.