Yersinia pestis in Pulex irritans Fleas during Plague Outbreak, Madagascar

To the Editor: Yersinia pestis (family Enterobacteriaceae) is a bacterium that can cause high rates of death in susceptible mammals and can provoke septicemic, pneumonic, and bubonic plague in humans (1). This zoonotic pathogen can be transmitted directly by infectious droplets or by contact with contaminated fluid or tissue or indirectly through flea bites (1).

Plague was introduced into Madagascar in 1898 from rat-infested steamships that had sailed from affected areas (2). Now, Madagascar is 1 of 2 countries in Africa that have reported cases of human plague every year since 1991 (3). During January 2008–January 2013, the number of human plague cases reported in Madagascar ranged from 312 to 648 per year. Of these, 61.8%–75.5% were laboratory confirmed (National Plague Laboratory of the Ministry of Health, pers. comm.). Most (>83%) confirmed cases were bubonic plague, which most commonly results from flea bites, suggesting that these bites were the most common mode of Y. pestis transmission. In Madagascar, Xenopsylla cheopis fleas have been known as the primary plague vector in urban areas, whereas Synopsyllus fonquerniei fleas have been usually involved in plague transmission in rural areas (2).

In January 2013, a total of 9 suspected bubonic plague cases, 3 confirmed, were reported in Soavina, a rural area in the district of Ambatofinandrana, Madagascar. Domestic fleas were collected with candle traps inside 5 houses during 3 nights (Table). Fleas were also caught on small mammals trapped inside houses and outside in the sisal fences and rice fields (Table). A total of 319 fleas belonging to 5 species in 5 genera were collected inside and outside the houses, an average of 44 per house (maximum 71): Pulex irritans, Echidnophaga gallinacea, and Ctenocephalides canis fleas were collected inside the houses (244, 76.5%), and S. fonquerniei and X. cheopis fleas were collected outside (75, 23.5%). The human flea, P. irritans, was the most collected flea species (233, 73.3%), followed by S. fonquerniei (62, 19.4%), X. cheopis (13, 4.1%), E. gallinacea (10, 3.1%), and C. canis (1, 0.3%).

Table. Fleas collected inside and outside houses in Soavina, Madagascar, January 2013.

Species	Total no. (%)	No. (%) inside	No. (%) outside
Pulex irritans	233 (73.0)	233 (95.5)	0
Ctenocephalides canis	1 (0.3)	1 (0.4)	0
Echidnophaga gallinacea	10 (3.1)	10 (4.1)	0
Synopsyllus fonquerniei	62 (19.4)	0	62 (82.7)
Xenopsylla cheopis	13 (4.1)	0	13 (17.3)
Total	319 (100)	244 (100)	75 (100)

Bacterial DNA was extracted from 277 fleas of 5 species: 233 P. irritans, 24 S. fonquerniei, 9 X. cheopis, 10 E. gallinacea, and 1 C. canis. PCR to detect Y. pestis was performed by using primers YP1 (5′-ATC TTA CTT TCC GTG AGA AG-3′) and YP2 (5′-CTT GGA TGT TGA GCT TCC TA-3′) to amplify a 478-bp fragment (4). Y. pestis DNA was then amplified and genotyped by Beckman Coulter Genomics Inc. (Takeley, United Kingdom). The positive control was Y. pestis reference strain (strain 6/69, 3 × 10⁸ bacteria/mL; Institut Pasteur de Madagascar).

Detection of Y. pestis was carried out on 274 fleas belonging to 5 flea species: 230 P. irritans (181 unfed and 49 engorged), 24 S. fonquerniei (15 unfed and 9 engorged), 9 X. cheopis (8 unfed and 1 engorged), 10 E. gallinacea (blood-feeding status not identified), and 1 unfed C. canis. Y. pestis was detected in 9 P. irritans fleas (7 male [6 unfed and 1 engorged] and 2 [engorged] female) from 3 houses, including the house where a confirmed human case of plague had occurred (Technical Appendix). Eight sequences (GenBank accession nos. KJ361938–KJ361945) were obtained and share 99% nucleotide homology with plasminogen activator genes of Y. pestis published in GenBank (accession nos. AF528537, AY305870). No Y. pestis was detected in the 24 S. fonquerniei, 9 X. cheopis, 10 E. gallinacea, or 1 C. canis fleas collected.

Although only X. cheopis and S. fonqueniei fleas had previously been described as plague vectors in Madagascar, P. irritans fleas were most commonly collected during this field study; engorged and unfed male and female P. irritans fleas carried Y. pestis. Other studies have found P. irritans fleas in the plague risk area in other countries in Africa (5,6); one study found that P. irritans fleas may play a role in plague epidemiology in Tanzania (5). Data on P. irritans fleas in rats make it unlikely that these fleas are involved in rat-to-human transmission of Y. pestis in Madagascar. During 1922–1995, a total of 118,608 rats were caught and examined in Madagascar, but only 148 P. irritans fleas were identified, and none have been found on rats since 1996 (http://www.pasteur.mg/spip.php?rubrique124). The high density of P. irritans fleas we observed in villages where plague outbreaks occurred in late 2012 and early 2013 (http://www.pasteur.mg/spip.php?rubrique124) supports the possibility that P. irritans fleas played a role in domestic human-to-human transmission of Y. pestis during these outbreaks.

Technical Appendix

Agarose gel electrophoresis showing positive PCR results for Yersinia pestis in DNA of 9 Pulex irritans fleas collected from 3 houses in an area of Madagascar where plague outbreaks occurred in late 2012 and early 2013.