Isolation of naturally infecting Leishmania infantum from canine samples in Novy-MacNeal-Nicolle medium prepared with defibrinated blood from different animal species.

This research article is investigating various types of animal blood to use for a medium that can grow the parasite Leishmania infantum, which can naturally infect dogs. This was done with the goal of finding alternative mediums to the standard which uses rabbit blood.

About the Study

• The objective of this study was to observe whether the parasite Leishmania infantum, sourced from naturally infected dogs, was viable in a culture medium made from the blood of different animal species.
• This was done in a region where visceral leishmaniasis, which is usually caused by L. infantum, is endemic.

The Medium and Sample Collection

• The medium used is a two-phase culture medium known as Novy-MacNeal-Nicolle (NNN) medium.
• The bloods of four different animal species (chicken, ox, horse, and sheep) were each used to prepare the NNN medium. These new media were labelled as NNN-C, NNN-O, NNN-H, and NNN-S correspondingly.
• The original medium prepared using rabbit blood was also used as a comparison and labelled as NNN-R.
• Spleen samples were taken from six dogs, which were all previously diagnosed as infected with the parasite.

Experiment and Results

• All of the collected samples were inoculated into each variant of the NNN medium. They were cultured and examined over a time period of three weeks under an optical microscope.
• Quantitative PCR (qPCR) was also used to analyze the spleen samples for parasite loads.
• Parasites from three of the dogs presented positive growth in at least one of the NNN test media. One of these three samples showed positive growth in all test mediums. The second sample resulted in the highest mean parasite count in the NNN-C medium, made from chicken blood. The third sample was positive only in the NNN-S medium, made from sheep blood, and in the standard control, NNN-R.
• For the remaining three dogs, the cultures in all NNN media, including the control and test media, presented no growth. Notably, two of these three dogs had the lowest spleen parasitic loads as per the results of the qPCR tests.
• The result of the experiment proved that chicken, horse, ox, and sheep blood were all viable for the preparation of NNN media for the primary isolation of L. infantum from the samples of naturally infected dogs.

Conclusion

• The bloods of the four animal species can be considered as alternatives to rabbit blood in the preparation of NNN culture medium, should the need arise.