The importance of subtype analysis of Cryptosporidium spp. in epidemiological investigations of human cryptosporidiosis in Iran and other Mideast countries.

Despite the clinical and public health importance of Cryptosporidium parvum, little is known about its transmission dynamics in cattle and other farm animals, especially in Iran and other Mideast countries. Currently, the maintenance of the parasites on cattle farms and the role of herd-to-herd transmission in cryptosporidiosis epidemiology are not clear (1).

Recent molecular epidemiologic studies of cryptosporidiosis have helped researchers to better understand the transmission of cryptosporidiosis in humans and the public health significance of Cryptosporidium spp. in animals and the environment (2, 3).

Because of the ability of Cryptosporidium spp. to infect humans and a wide variety of animals, and because of the ubiquitous presence of Cryptosporidium oocysts in the environment, humans can acquire Cryptosporidium infections through several transmission routes, such as direct contact with infected persons (person-to-person transmission) or animals (zoonotic transmission), and ingestion of contaminated food (foodborne transmission) and water (waterborne transmission). The relative importance of these transmission routes in the epidemiology of cryptosporidiosis is not entirely clear, largely due to the fact that traditional diagnostic tools do not have the ability to differentiate sources of parasites (3).

The use of molecular tools has been helpful in assessing the zoonotic potential of various Cryptosporidium species and the sources of human infection; it has begun to play a significant role in the characterization of transmission dynamics in different areas and in the determination of host specificity of various Cryptosporidium spp. The 60 kDa glycoprotein (gp60, also known as Cpgp15/45) gene encodes a precursor protein that is proteolytically cleaved to yield mature surface glycoproteins gp45 and gp15 (also known as Cp17), both of which are implicated in the attachment and invasion of enterocytes by sporozoites and merozoites.

An important feature of this gene is its high degree of sequence polymorphism in C. hominis, C. parvum, and C. meleagridis isolates. Several subtype families have been identified in these species: 7 subtype families in C. hominis (Ia–Ig), 2 zoonotic (IIa, IId) and 10 non-zoonotic (IIb, IIc, IIe–IIl) subtype families in C. parvum, and 6 subtype families in C. meleagridis (4). Within each subtype family, there are multiple subtypes based primarily on the number of tri-nucleotide repeats coding for the amino acid serine, as suggested by Sulaiman et al. (2005) (5).

The use of gp60 subtyping has allowed the identification of geographic and temporal differences in the transmission dynamics of cryptosporidiosis, the role of zoonotic infections in epidemiology, better appreciation of the public health significance of Cryptosporidium species/genotypes in ruminants and significance of parasite subtypes/strains in clinical manifestations and outbreak potentials, and the tracking of infection and contamination sources during outbreak and endemic investigations (1–5).

To our knowledge, there are several molecular epidemiological studies that have documented the presence of C. parvum and C. hominis In Iran (Table 1) (6–12). However, the distribution of subtypes of the two species in humans, animals and environmental is unclear. In the first characterization of Cryptosporidium subtypes in humans and cattle in Iran by sequence analysis of the gp60 gene, 47 samples of C. parvum (22 from children and 25 from cattle) and three of C. hominis (all from children) were characterized. Nine subtypes (two of C. hominis and seven of C. parvum) belonging to four subtype families were found. Cattle were mainly infected with C. parvum IIa subtypes and humans mostly with C. parvum IIa and IId subtypes (Table 2). The predominance of IIa and IId subtypes underlines the importance of zoonotic Cryptosporidium transmission in Iran. Thus, cattle could be a source of human infection with C. parvum IIa in Iran (13–15). Although the source of IId subtypes in humans is not yet clear, IId subtypes are known to be common in sheep and goats in some countries such as Spain (16) and in dairy cattle in some other countries such as Egypt (17) and China (18). Further molecular study in humans and animals is needed in order to determine the extent and animal source of zoonotic transmission of cryptosporidiosis in Iran.

Table 1

Distribution of Cryptosporidium spp. in humans in Mideast countries

Population	N	C. hominis	C. parvum	C. meleagridis	C. felis	C. canis	Mixed species	Reference
Iran	15	4	12					(9)
Iran	24	17	7					(10)
Iran	21	15	6					(11)
Iran	25	3	22					(12)
Turkey	4		4					(19)
Kuwait	62	3	58				2	(5)
Kuwait	83	22	61					(20)
Jordan	44	20	22	1				(21)
Saudi Arabia	31	13	15	1			1	(22)
Saudi Arabia	53	9	43				1	(23)
Egypt	36	24	10	2				(24)
Egypt	15	9	3					(25)

Table 2

Distribution of C. parvum subtypes in humans and cattle in Iran.

subtype families	Subtype	No. of isolates	Source of Samples	Accession number
IIa	IIa A16G3R1	1	Cattle	AB560739
IId	IId A15G1	2	Cattle	AB560740
IIa	IIa A15G2R1	22	Cattle	AB560741
IId	IId A26G1	1	Children with diarrhea	AB560742
IId	IId A18G1	3	Children with diarrhea	AB560743
IIa	IIa A16G3R1	1	Children with diarrhea	AB560744
IId	IId A20G1a	9	Children with diarrhea	AB560745
IId	IId A21G1a	1	Children with diarrhea	AB560746
IIa	IIa A15G2R1	6	Children with diarrhea	AB560747
IId	IId A15G1	1	Children with diarrhea	AB560748

The dominance of C. parvum and wide occurrence of IId C. parvum subtypes in humans in Iran is similar to the situation seen in other Mideast countries (9–12, 19–25) (Tables 1 and 3). Children in the Kuwait City are almost exclusively infected with IIa and IId subtypes, although they have little contact with farm animals. As the city uses desalinized sea water as drinking water, the C. parvum transmission appears to be anthroponotic in origin (5, 20). IId subtypes are also common in children in Saudi Arabia and Jordan (Table 3). In many industrialized nations in other areas, C. parvum infections are much less common in humans than C. hominis infections, with the exception of European countries and New Zealand, where both C. parvum and C. hominis are commonly seen in humans. In these industrialized nations, most C. parvum infections are caused by the IIa subtypes commonly found in cattle, indicating zoonotic transmission plays a significant role in cryptosporidiosis epidemiology. In contrast, humans in developing countries are much less commonly infected with C. parvum and most of the few C. parvum infections are caused by the anthroponotic IIc subtype family (2).

Table 3

Distribution of C. parvum subtype families in humans in Mideast countries

Location	N	Subtype Family						Reference
		IIa	IIc	IIb	IId	IIe	Other
Kuwait	59	28	2	0	29	0	1	(5)
Kuwait	61	29	10	0	19	0	3	(20)
Saudi Arabia	37	1	2	0	34	0	0	(23)
Jordan	13	3	2	0	8	0	0	(21)
Iran	22	7	0	0	15	0	0	(13, 14, 15)

In conclusion, preliminary molecular epidemiological studies have revealed some unique features of cryptosporidiosis transmission in humans in Iran and other Mideast countries. As the C. parvum subtype family IId was the dominant family causing cryptosporidiosis in humans in Iran (13–15), zoonotic transmission could possibly be involved. However, more extensive sampling of both humans and farm animals, especially sheep and goats, and collection of epidemiological data in case-control and longitudinal studies are needed for a better understanding of the sources of C. parvum infections in humans in Iran and other Mideast countries.