Prevalence and Genetic Diversity of Bartonella Species Detected in Different Tissues of Small Mammals in Nepal

Abstract

Bartonellae were detected in a total of 152 (23.7%) of 642 tissues from 108 (48.4%) of 223 small mammals trapped in several urban areas of Nepal. Based on rpoB and gltA sequence analyses, genotypes belonging to seven known Bartonella species and five genotypes not belonging to previously known species were identified in these animals.

Bartonella species are important zoonotic and vector-borne bacteria associated with an increasing array of emerging infections in humans and animals (6, 8, 21). Identification of new bartonellae, especially in animals that have possible contact with humans, can help to identify new human pathogens (12). Moreover, the close association between rodents and humans throughout the world makes the study of rodent-borne Bartonella essential to determine the extent to which rodents may serve as a source of human infections (7). We conducted a study on the molecular detection of Bartonella in different tissues of Nepalese small mammals.

In 1996, a total of 223 small mammals (38 Bandicota bengalensis, 3 Mus musculus castaneus, 90 Rattus rattus brunneusculus, and 92 Suncus murinus animals) were trapped in densely human populated urban areas in Nepal. Details on the trapped animals are given in Table 1. Lung, kidney, and liver tissue samples were collected and shipped to the Centers for Disease Control and Prevention (CDC), Fort Collins, CO. A total of 642 tissue samples were assessed for the presence of Bartonella DNA by rpoB and gltA sequencing as described previously (5, 23).

TABLE 1.

Prevalence of Bartonella in small mammals trapped in different districts of Nepal

Districta	Trap areab	No. of Bartonella-infected animals/no. of animals examined (%)
		Bandicota bengalensis	Mus musculus castaneus	Rattus rattus brunneusculus	Suncus murinus	Subtotal	Total
Bhaktapur	Bhelukhel			3/5 (60)		3/5 (60)
	Bhelukhel			1/1 (100)		1/1 (100)
	Pode Tole	0/2 (0)		4/8 (50)	0/1 (0)	4/11 (36.4)
	NK			4/12 (33.3)	4/9 (44.4)	8/21 (38.1)
Subtotal		0/2 (0)	0/0 (0)	12/26 (46.2)	4/10 (40)		16/38 (42.1)
Kathmandu	Hyumata			2/2 (100)	2/3 (66.7)	4/5 (80)
	Inakha Tole				3/3 (100)	3/3 (100)
	Kalimati	2/6 (33.3)	0/1 (0)	1/5 (20)	7/12 (58.3)	10/24 (41.7)
	Lagan Tole	0/4 (0)		2/3 (66.7)	3/5 (60)	5/12 (41.7)
	Sabalbahal	7/21 (33.3)	0/1 (0)	2/6 (33.3)	7/14 (50)	16/42 (38.1)
	Teku	1/4 (25)	0/1 (0)	4/13 (30.8)	1/1 (100)	6/19 (31.6)
	Tokha (suburb)			1/1 (100)		1/1 (100)
	NK			1/2 (50)	15/25 (60)	16/27 (59.3)
Subtotal		10/35 (28.6)	0/3 (0)	13/32 (40.6)	38/63 (60.3)		61/133 (45.9)
Lalitpur	Bakhar			4/4 (100)		4/4 (100)
	Bakhar Thati			0/3 (0)		0/3 (0)
	Lagankhel			1/1 (100)		1/1 (100)
	Patan				2/2 (100)	2/2 (100)
	Sabalbahal	0/1 (0)				0/1 (0)
	NK			9/24 (37.5)	15/17 (88.2)	24/41 (58.5)
Subtotal		0/1 (0)	0/0 (0)	14/32 (43.8)	17/19 (89.5)		31/52 (59.6)
Total		10/38 (26.3)	0/3 (0)	39/90 (43.3)	59/92 (64.1)		108/223 (48.4)

^aThe following Bartonella species were detected: in Bhaktapur, B. coopersplainsensis, B. elizabethae, B. queenslandensis, and a novel Bartonella species; in Kathmandu, B. coopersplainsensis, B. elizabethae, B. queenslandensis, B. rochalimae, B. tribocorum, and a novel Bartonella species; and in Lalitpur, B. coopersplainsensis, B. elizabethae, B. phoceensis, B. queenslandensis, B. rattimassiliensis, B. tribocorum, and a novel Bartonella species.

^bNK, not known.

Bartonella was detected in 108 (48.4%) of 223 animals in Nepal: 10 (26.3%) of 38 B. bengalensis rats, 39 (43.3%) of 90 R. rattus brunneusculus rats, and 59 (64.1%) of 92 S. murinus shrews were infected with Bartonella (Table 1). The infection rate varied from 42 to 60% by province. The highest numbers of infected animals were in the Kathmandu and Lalitpur districts. This was because S. murinus was the most commonly infected species and the highest numbers of animals of this species were trapped in these areas. Bandicoots were the most commonly infected subjects in Kathmandu, but fewer numbers of these animals (n = 2) were trapped in Bhaktapur and Lalitpur. Only three M. musculus mice were collected (only in Kathmandu) in this study, and none of these animals were positive for Bartonella. A total of 152 (23.7%) of 642 tissue samples were positive for Bartonella. Of the three tissue types, kidney samples (29.6%) and liver samples (25.6%) were more frequently infected with Bartonella than the lung samples (17.3%; P = 0.03 and 0.04, respectively) (Table 2). DNA sequencing revealed 37 and 20 rpoB and gltA genotypes, respectively, of which 31 and 15 belonged to B. rattimassiliensis, B. queenslandensis, B. elizabethae, B. tribocorum, “B. rochalimae,” or “B. phoceensis” and the other 6 and 5 genotypes, respectively, were not genetically related to known bartonellae (Fig. 1 and 2). These novel genetic groups were found in S. murinus and R. rattus brunneusculus animals. The human pathogen B. elizabethae was detected in R. rattus brunneusculus rats (n = 5) and an S. murinus shrew (n = 1), and the human pathogen B. rochalimae was found in a B. bengalensis bandicoot (n = 1) and an S. murinus shrew (n = 1).

TABLE 2.

Detection of Bartonella DNA in different tissues of small mammals in Nepal

Species	No. of animals infected/no. of animals tested (%)	No. of Bartonella-infected tissue samples/no. of tissue samples examined (%)
		Kidney	Liver	Lung	Total
Bandicota bengalensis	10/38 (26.3)	5/38 (13.2)	2/38 (5.3)	5/18 (27.8)	12/94 (12.8)
Mus musculus castaneus	0/3 (0)	0/3 (0)	0/3 (0)	0/3 (0)	0/9 (0)
Rattus rattus brunneusculus	39/90 (43.3)	12/90 (13.3)	32/90 (35.6)	15/89 (16.9)	59/269 (21.9)
Suncus murinus	59/92 (64.1)	49/92 (53.3)	18/92 (19.6)	14/86 (16.3)	81/270 (30)
Total	108/223 (48.4)	66/223 (29.6)	52/203 (25.6)	34/196 (17.3)	152/642 (23.7)

FIG. 1.

Phylogenetic classification of Bartonella genotypes detected in small mammals of Nepal based on rpoB gene sequences. The phylogram was constructed by the neighbor-joining method with the Kimura 2-parameter model. Only the bootstrap values above 70% obtained from 1,000 replicates are given. The 76 different sequences of rpoB from small mammals of Nepal were classified into seven clusters. The GenBank accession numbers for reference sequences are given in parentheses. Only known reference Bartonella sequences without gaps and missing data are included in the phylogram. The rpoB gene sequence of Brucella melitensis 16M^T was included as an out-group.

FIG. 2.

Phylogenetic classification of Bartonella genotypes found in small mammals of Nepal based on gltA gene sequences. The phylogram was constructed by the neighbor-joining method with the Kimura 2-parameter model. Only the bootstrap values above 70% obtained from 1,000 replicates are given. The 37 different sequences of gltA from small mammals of Nepal were classified into seven clusters. The GenBank accession numbers for reference sequences are given in parentheses. Only known reference Bartonella sequences without gaps and missing data are included in the phylogram. The gltA gene sequence of Brucella melitensis 16M^T was included as an out-group.

Our study reports the first molecular detection and findings on the prevalence of Bartonella in small mammals of Nepal and is also the first study to compare different organ tissues from the same animal for Bartonella detection. Previously, several reports were made on the occurrence of bartonellae in rodents and other small mammals in Asian countries, including Bangladesh (3), China (28), Indonesia (27), Japan (16), Laos (1), Taiwan (19), and Thailand (2, 7, 24). Recently, Bai et al. (3) reported that 63.2% of B. bengalensis, 32.3% of R. rattus, and 42.9% S. murinus animals collected in Dhaka, Bangladesh, were infected with Bartonella, and the isolates from these animals were genetically related to Bartonella isolates found in America and Europe. Our study produced similar results in terms of the infectivity of the animals and the specificity of bartonellae. It is evident from all these studies that B. elizabethae, B. phoceensis, B. rattimassiliensis, B. rochalimae, and B. tribocorum are being circulated among small animals in Asia. Furthermore, B. coopersplainsensis and B. queenslandensis, found initially in Australia, were also detected in Nepal (this study) and Bangladesh (3), suggesting that the origin of these species might be Australasia.

The prevalence of Bartonella in Nepal was comparable with prevalence rates on other continents: in Africa, 44% in South Africa (22) and 24% in the Democratic Republic of Congo and Tanzania (V. A. K. B. Gundi et al., unpublished data); 42% in North America (18); and in Europe, 17% in France (12), 17% in central Sweden (14), 28% in southwestern Spain (20), 28% in Denmark (11), 31% in Greece (25), 31% in northeast Poland (26), 40% in Slovenia (17), and 64% in the United Kingdom (4). In a recent study, 29% prevalence was recorded in Australia (13). Such high rates of Bartonella prevalence are significant in the context of human health, because the synanthropic mammals harboring pathogenic microorganisms are often found in biotopes where they can come into close contact with humans, who might therefore be at some risk of exposure.

In this study, specificity of some bartonellae for host animals was observed (Table 3). The R. rattus brunneusculus rats carried almost all the rat-origin Bartonella species detected in this study, except B. rochalimae, which was isolated recently from R. norvegicus in Taiwan (19). In our study, S. murinus shrews harbored mostly B. queenslandensis. Studies by Bai et al. conducted in Dhaka, Bangladesh (3), also yielded evidence of the host specificity of B. queenslandensis for S. murinus. In addition, S. murinus carried some novel bartonellae in our study. On the other hand, S. murinus did not harbor other bartonellae, such as B. coopersplainsensis, B. phoceensis, and B. rattimassiliensis. These findings appear to suggest specificity of the bacteria for the host.

TABLE 3.

Bartonella coinfections in Nepalese small mammals

Host species	No. of animals	District	Areaa	Cosurviving Bartonella combination	Genotype(s) for:
					rpoB	gltA
Bandicota bengalensis	1	Kathmandu	Kalimati	B. coopersplainsensis and B. queenslandensis	35, 11
Rattus rattus brunneusculus	1	Lalitpur	NK	B. coopersplainsensis and B. phoceensis	33, 27	20
	1	Lalitpur	NK	Novel Bartonella sp. and B. rattimassiliensis	29, 30, 31	1, 2
	1	Lalitpur	NK	B. coopersplainsensis and B. rattimassiliensis	34	3
	1	Lalitpur	NK	B. elizabethae and B. tribocorum	24	9
Suncus murinus	10	Kathmandu	Hyumata	B. queenslandensis and novel Bartonella sp.	13	14
		Lalitpur	Patan		1	15, 17
		Kathmandu	Sabalbahal		10	17
		Kathmandu	Sabalbahal		13	14
		Kathmandu	NK		15, 36	17
		Kathmandu	NK		1	16
		Kathmandu	NK		2	15
		Kathmandu	NK		2	15
		Kathmandu	NK		13	14
		Lalitpur	NK		16	17
	1	Kathmandu	Sabalbahal	B. queenslandensis and B. rochalimae	13	18
	1	Lalitpur	NK	B. elizabethae and novel Bartonella sp.	23, 36
Total	17

^aNK, not known.

The prevalence and diversity of Bartonella species in animals or other hosts are controlled by geographical location, environmental conditions, the presence of vectors, and host species and habitats. In our study, 17 animals (15.7%) were infected with multiple Bartonella species and most of these animals had dual infections with different combinations of species (Table 4). This may be due to vector transmission between different animal hosts. According to Ellis and others, Bartonella species associated with hosts native to the Old World are phylogenetically distinct from those associated with hosts native to the New World (10). In this study, however, genotypes belonging to B. elizabethae, B. phoceensis, B. rattimassiliensis, and B. tribocorum were closely related to the isolates found in America and Europe (Tables 5 and 6), suggesting that carriers of these species might have migrated from Asia to other continents. Moreover, some novel genomic groups detected in this study were 98.3 to 100% identical to Bartonella strains found in Africa (Gundi et al., unpublished data) (Table 5). Furthermore, some genotypes, including novel genomic groups, from Nepalese rodents and shrews clustered well with other well-known rodent-associated species and human pathogens, such as B. coopersplainsensis, B. elizabethae, B. phoceensis, B. queenslandensis, B rattimassiliensis, B. rochalimae, and B. tribocorum (Fig. 1 and 2). These findings suggest that small mammals in Nepal, like those in other Asian countries, might be a potential source of Bartonella infections. Furthermore, these results suggest the need to conduct further studies to verify whether these agents might be responsible for human cases of febrile illness of unknown etiology and to determine the evolutionary, genetic, and pathogenic relationships among Nepal isolates and other isolates in Asia and on other continents.

TABLE 4.

Association between Bartonella genotypes and mammalian hosts

Bartonella species	No. of animals associated with specific Bartonella organism(s)
	Bandicota bengalensis	Rattus rattus brunneusculus	Suncus murinus	Mus musculus castaneus	Total
B. coopersplainsensis	1	3			4
B. elizabethae		5	1		6
B. phoceensis		1			1
B. rattimassiliensis		3			3
B. rochalimae	1		1		2
B. tribocorum	1	4	2		7
B. queenslandensis	1	2	47		50
Novel Bartonella sp.		3	12		15
Combination of species	1	4	10		15
Total no. of associated animals/total no. of animals	5/38	25/90	73/92	0/3	103/223

TABLE 5.

Genetic relationships between Bartonella species detected in Nepal and those from other geographical regions based on rpoB sequence analysis

Bartonella sp. identified	Genotype(s) determined in this study	Host animal(s) in this studya	Accession no. of related GenBank sequence	% Sequence similarity	Host(s) of related sequence	Country of host(s)	Referenceb
B. coopersplainsensis	32	RRB	EU714973	100	Rattus rattus	Laos	1
			EU111792	100	Rattus leucopus	Australia	13
B. phoceensis	27	RRB	AY515132	99.5	Rattus norvegicus	France	12
			AB290278	99.5	Rattus rattus	Japan	16
Novel Bartonella sp.	28	RRB	FJ667575	98.8	Unknown	Taiwan	D. D. Ji et al., unpublished data
B. queenslandensis	1-19	SM	EF204538	98.1-99.3	Suncus murinus	Bangladesh	3
			EF204539	97.2-100			3
			AB290268	97.2-100	Unknown	Japan	16
			AB290269	97.1-99.9	Unknown
			AB290270	97.6-98.8	Rattus rattus
	20	RRB	AB290266	100	Rattus rattus	Japan	16
			EU111787	98.4	Melomys sp.	Australia	13
			EU111788	99.2	Rattus tunneyi
			EU111789	98.8	Rattus fuscipes
			EU111790	99.5	Rattus conatus
			EU111791	99.3	Rattus leucopus
Novel Bartonella sp.	23	SM	FJ851123	98.3	Lophuromys rita	Democratic Republic of Congo	Gundi et al., unpublished
			FJ851124	100	Mus minutoides
			FJ851125	100	Mus minutoides
			FJ851126	100	Mus minutoides
			FJ851129	100	Mus minutoides
			FJ851144	98.3	Lophuromys sp. (Lophuromys dudui related)	Tanzania	Gundi et al., unpublished
			FJ851146	98.2	Lophuromys sp. (Lophuromys dudui related)
B. tribocorum	22	BB, RRB	EU714974	99.9	Rattus rattus, Mus cervicolor, Cannomys badius, Rattus exulans	Laos	1
			EU714975	99	Unknown
B. elizabethae	24	RRB	AF165992	98.3	Unknown	France	23
			FJ851128	98.1	Arvicanthis neumanni	Democratic Republic of Congo	Gundi et al., unpublished
B. rochalimae	37	BB	EU551156	99.6	Rattus norvegicus	Taiwan	19

^aBB, Bandicota bengalensis; RRB, Rattus rattus brunneusculus; SM, Suncus murinus.

^bThe unpublished data cited are found in the GenBank database.

TABLE 6.

Genetic relationships between Bartonella species detected in Nepal and those from other geographical regions based on gltA sequence analysis

Bartonella sp. identified	Genotype determined in this study	Host animal(s) in this studya	Accession no. of related GenBank sequence	% Sequence similarity	Host of related sequence	Country of host	Referenceb
B. rattimassiliensis	1	RRB	FJ179375	100	Rattus norvegicus	Taiwan	19
			AY515125	100	Rattus norvegicus	France	12
	3	RRB	AB290283	100	Rattus rattus	Japan	16
			AF342933	100	Rattus fuscipes	China	28
			FJ464242	100	Rattus sp.	China	D. Li et al.,
			FJ464244	100	Rattus sp.		unpublished data
			FJ492787	100	Rattus tanezumi
			FJ492792	100	Rattus tanezumi
			FJ589047	100	Rattus tanezumi
B. elizabethae	7	RRB	AY589561	100	Bandicota bengalensis	Bangladesh	3
	8	RRB	AB445000	100	Acomys cahirinus	Japan	15
			DQ884386	100	Rattus norvegicus	China	Li et al., unpublished
			DQ884390	100	Rattus norvegicus	China	Li et al., unpublished
			FJ655404	100	Rattus exulans	Thailand	2
			FJ946849	100	Dog	Thailand	Y. Bai et al., unpublished data
			U28072	100	Unknown	Unknown	D. C. Jones et al., unpublished data
			Z70009	100	Human	United States	9
			Z70020	100	Rattus sp.	Peru	5
	6	RRB	AF329679	100	Rattus fuscipes	China	28
			FJ179377	100		Taiwan	J. W. Hsieh et al., unpublished data
			FJ179391	100		Taiwan	Hsieh et al., unpublished
			FJ492786	100	Rattus tanezumi	China	Li and Liu, unpublished
			FJ492791	100	Rattus tanezumi
			FJ492793	100	Rattus tanezumi
			FJ492797	100	Rattus tanezumi
			FJ589045	100	Rattus tanezumi	China	Li et al., unpublished
			FJ589046	100	Rattus tanezumi
			FJ589050	100	Rattus tanezumi
			FJ589052	100	Rattus tanezumi
			FJ589053	100	Rattus tanezumi
			FJ589058	100	Rattus tanezumi
			FJ589061	100	Rattus tanezumi
			FJ589062	100	Rattus tanezumi
B. queenslandensis	5	RRB	AB290280	100	Rattus rattus	Japan	16
			AY589566	100	Rattus rattus	Bangladesh	3
			FJ179376	100		Taiwan	19
			FJ179384	100
			FJ946846	100	Dog	Thailand	Bai et al., unpublished
B. tribocorum	13	RRB, BB	AF086636	100	Rattus rattus	Portugal	10
	10	RRB, BB	AF075164	100	Rattus norvegicus	United States	10
			AJ583111	100	Unknown	South Africa	22
			AY902183	100	Rattus tanezumi	Indonesia	27
			AY902188	100
			AY902189	100
			AY902191	100
			DQ884383	100	Rattus norvegicus	China	Li et al., unpublished
			DQ884387	100
			DQ884388	100
			DQ884389	100
			DQ884391	100
			DQ884392	100
			EF051466	100	Rattus norvegicus	China	Li et al., unpublished
			FJ464203	100	Rattus norvegicus	China	Li et al., unpublished
			FJ464205	100
			FJ464206	100
			FJ492782	100	Rattus tanezumi	China	Li and Liu, unpublished
			FJ492783	100	Rattus tanezumi
			FJ492785	100	Rattus tanezumi
			FJ492789	100	Rattus tanezumi
			FJ492794	100	Rattus tanezumi
			FJ492795	100	Rattus tanezumi
			FJ492798	100	Rattus tanezumi
			FJ492800	100	Rattus tanezumi
			FJ589051	100	Rattus tanezumi	China	Li et al., unpublished
			FJ589057	100	Rattus tanezumi
B. rochalimae	18	SM	EU551154	100	Rattus norvegicus	Taiwan	19

^aBB, Bandicota bengalensis; RRB, Rattus rattus brunneusculus; SM, Suncus murinus.

^bThe unpublished data cited are found in the GenBank database.

Nucleotide sequence accession numbers.

The DNA sequences obtained in this study were deposited in GenBank under accession numbers GU143433 to GU143508 (for rpoB) and GU143509 to GU143549 (for gltA) (see Tables S1 and S2 in the supplemental material).