Low prevalence of Neospora caninum and Toxoplasma gondii antibodies in dogs in Jilin, Henan and Anhui Provinces of the People’s Republic of China

Neospora caninum and Toxoplasma gondii are related coccidians that until 1988 were considered the same organism [1]. N. caninum is now considered the most important abortifacient for cattle worldwide, including China [2],[3]. Dog, wolf, coyote and dingo are the definitive hosts for N. caninum that shed environmentally resistant oocysts. Toxoplasma gondii infection in dogs is important for following reasons. Dogs can be infected through contact with the T. gondii, which may be acquired from rooting in infected soil or from ingesting cat feces or from eating raw meat. Dogs can also mechanically transmit T. gondii oocysts to humans [4]. In China, dogs serve as food animals, the consumption of undercooked meat containing T. gondii tissue cysts can be a supplementary health risk to consumers.

Currently, there is no report of isolation T. gondii from dog in China, and little is known of N. caninum infections in dogs in China. The objective of present study was to determine the seroprevalence of N. caninum and T. gondii infections in dogs from China, and to attempt isolate T. gondii.

Six (6.25%) of 96 dogs were seropositive of N. caninum with titers of 1:25 in 2, 1:50 in 3, and 1:100 in 1, and all seropositive dogs were more than 1 year old (Table 1). Antibodies to N. caninum were found in 6 (13.95%) of 43 dogs from Jilin but not in 53 dogs from the Henan and Anhui, because most of the dogs from other two regions were less than one year old. Antibodies to T. gondii were found in 35 of 425 (8.24%) dogs with titers of 1:25 in 15, 1:50 in 14; and 1:100 in 6, none was positive at 1:200 (Table 1). The age range of the N. caninum and T. gondii negative dogs was 1.06 ± 0.55 (years) and 2.90 ± 1.38 (years). The age range of the N. caninum and T. gondii positive dogs was 2.25 ± 1.42 (years) and 3.06 ± 2.16 (years). T. gondii was not isolated in mice inoculated with tissues of any dogs.

Hearts samples and serum were kept cold and transported by air from China to USA within one week of killing. One week antibodies are fairly stable. T. gondii antibodies were stable on dried filter papers for up to 45 days at room temperature [8] and for 6 months at 25°C when stored with silica gel [9]. In an unpublished experiment antibodies were still detectable in blood 7 days after they were accidently left in a water bath at 37°C (JP Dubey, unpublished 1971).

In the present study, antibodies to N. caninum were detected in dog sera for the first time from China. How dogs become infected with N. caninum in nature is unknown [1]. Congenital transmission and ingestion of infected tissues or oocysts are the three probable modes of transmission. Of these, the ingestion of infected tissues is considered the most important because congenital transmission in dogs is relatively rare and fecal transmission has not been proven [3]. Little is known of the natural epidemiology of N. caninum in nature. In the USA, cattle and deer are commonly infected with N. caninum. Viable parasite has not been isolated from small mammals. Little is known of the epidemiology of neosporosis in China. In this study, antibodies to N. caninum were 6.25% (6/96) of first batch samples tested. We didn’t test the second batch samples because of non availability of reagents for testing of N. caninum by NAT, or IFA, or cELISA in China. Compared with other countries [10]-[13], the prevalence of N. caninum antibody was low in East Asia [14],[15]; also it is low in this study. Although a comparison of different serological tests (NAT, IFA, cELISA) for the detection of N. caninum antibodies in dogs has not been made, it is generally accepted that results by NAT and IFA are comparable because both tests assess antibodies directed against whole tachyzoites [1]. None of the serological tests, however, have been validated because it is very difficult to isolate N. caninum from asymptomatic hosts [1].

In this study, the prevalence of antibodies and titers to T. gondii were low compared to data from other parts of China (Table 2, Figure 1) and other country [16],[17]. However, results are not strictly comparable because of different serological tests used and different cut-off used. In several of these previous reports, the serological tests used have not been critically evaluated. To facilitate further investigations we have summarized available reports on canine toxoplasmosis in Table 2. The MAT we used has been extensively used for the detection of T. gondii antibodies in many species, including humans and dogs [7]. Viable T. gondii was isolated from 51% (22/43) of dogs with MAT antibodies [18] and there is no evidence for any cross reactivity with other antigens in MAT [19]. The lack of isolation of T. gondii from the hearts of any of the dogs in the present study could be related to the loss of infectivity during transit from China and USA. However, viable T. gondii has been isolated in the USDA laboratory in Beltsville from tissues of animals that had been in transit for up to 10 days from several countries to USA. The survival of T. gondii tissue in meat at room temperature is largely unknown. However, viable T. gondii was isolated from a Hawaiian bird that was completely rotten and most carcasses had been eaten by maggots [20]. At 40°C, T. gondii tissue cysts survived up to 2 months [21]. Temperatures at which the dog tissues samples were stored varied from 4-20°C while in transit from China to USA. Therefore, we are uncertain of the effect of storage on the survival of tissue cysts.

Map region	Provinces/cities	Year tested	Dog type	No. tested	No. positive	% positive	Serologic test	No. with terminal titer of					Reference
								<25	25 ~ 49	50 ~ 99	100 ~ 199	≥200
I	Beijing	1999-2005	Pet	534	128	24.0	ELISAa/LATb	unknown					[22]
II	Heilongjiang	2010-2011	Stray/pet	124	14	11.3	IHAb	unknown					[23]
III	Liaoning	2012	Pet	328	33	10.0	IHAb	unknown					[24]
		2012	Police	291	90	30.9	MATe	45	27	0	12	6	[25]
IV	Shangdong	2010-2011	Stray/pet	143	14	9.8	IHAb	unknown					[23]
V	Henan	2010-2011	Stray/pet	106	13	12.3	IHAb	unknown					[23]
		2013	Countryside	31	1	3.2	MATe	1	1	0	0	0	Present study
VI	Jiangshu	unknown	Stray/countryside	231	93	40.3	ELISAa	unknown					[26]
		2010	Household	288	62	21.5	MATe	21	15	0	11	9	[27]
VII	Shanghai	2009	City pet	1178	15	1.3	IHAb	unknown					[28]
			Town pet	364	22	6.0
			Countryside	194	19	9.8
VIII	Xinjiang	2010-2011	Stray/pet	259	29	11.2	IHAb	unknown					[23]
IX	Gansu	2010	Pet	259	28	10.8	MATe	14	9	4	1	0	[29]
X	Sichuan	2010	Household	314	11	3.5	IHAb	0	0	4	3	4	[30]
XI	Yunnan	2011-2012	Pet	611	132	21.6	IHAc	0	0	29	25	78	[31]
XII	Guangdong	unknown	Stray	36	12	33.3	ELISAd	unknown					[32]
			Household	114	20	17.5
XIII	Jilin	2013	Countryside	43	7	16.3	MATe	5	6	1	0	0	Present study
		2014	Countryside	329	27	8.2		0	8	13	6	0
XIV	Anhui	2013	Countryside	22	0	0	MATe	0	0	0	0	0	Present study

IHA: indirect hemagglutination test, ELISA: enzyme-linked immunosorbent assay, LAT: latex agglutination test.

^a:ELISA Kit produced by Zhuhai S.E.Z Haitai Pharmaceutlcals Co., Ltd, China.

^b:Kit produced by Lanzhou Veterinary Institute, Academy of Agriculture and Science, China.

^c:Kit produced by Nanjing Veterinary research Institute, Jiangshu Academy of Agriculture and Science, China.

^d:Kit produced by Combined Company, Shenzhen, China.

^e:in-house.

Results of the present study indicated low prevalence of N. caninum and T. gondii infection in dogs in China, compared with data from Europe and America. Identification of the risk factors that underlie these differences may help prevention of neosporosis and toxoplasmosis. Dog meat is consumed by local people in China. Therefore, dogs are at a risk of T. gondii infection and should be of public health concern.