Jpn. J. Infect. Dis., 54, 23‑26, 2001

Original Article

Prevalence of Cpyptosporidium parvum Infections in Weaned Piglets andFattenlng Porkers in Kanagawa Prefecture, Japan

Shinji Izumlyama, Ichiro Furukawal , Toshiro Kurokil , Shiro Yamail ,Hiromu Sugiyama2, Kenji Yagita2 and Takuro Endo2*
Tsukuba ITechnical Center, Ikeda Scientljic Co. Ltd.,

Oaza‑Akatsuka Aza‑Ushigafuchi 586‑9, Tsukuba, Ibaraki 305‑0062,
lDepartment of Bacteriology and Pathology, Kanagawa Prefectural Public Health Laboratory,
Naka0‑machi 52‑2, Asahi‑ku, Yokohama, Kanagawa 241‑0815 and2Departmen t ofParasitology, National Institute ofInfectious Diseases,

Toyama 1‑23‑1, Shinjuku‑ku, Tob}0 162‑8640, Japan(Received February 15, 2001. Accepted March 8, 2001)
SUMMARY: Fecal samples fTrom 232 weaned piglets (1 and 3 months old) and 252 fattening porkers (6 monthsold) in 8 stock‑raising farms located in Kanagawa Prefecture, Japan,斤om June 1 998 to June 2000 Wでre examined

to determine the prevalence of Cywtosporidium infection. Detection of oocysts was performed uslng the ethyl
acetate fecal concentration method and immunofluorescent stainlng. C. pamm oocysts were identified in 77(33.2%) 1‑3 months old weaned pigletsfrom four farms. The odds of excreting among 1‑3 months old piglets
were more than 100 times greater thanチmong 6 months old porkers (95% confidence interval‥ 17‑902)･ Thisstrongly suggests that weaned pigletsare Important reservoirs of pathogenic microbes whose potential contamina‑tion of drinking water has epidemiological implications for human health.

various locations worldwide. However, due to the generally

INTROD UC TION

accepted oplnion that C. parvum is not a serious cause of

The increaslng WOrld population has led to increaslng Beshenteritis in either neonatal or weaned piglets, only a limited

water requlrementS for drinking, hygiene and household

number of epidemiologlCal studies have yet been reported,

purposes. Population growth also brings ln its train highloads

making it di瓜cult toknow the prevalence ofinfectioninmanyof munlCIPal sewage, livestock excreta and industrial waste‑countries including Japan.Asdistinct from the case ofwater which threaten the sources of drinking water. While

cryptosporidiosisinruminants, whereininfeCtion lS generallywater treatment technology can successfully process water

established during the first 2 weeks after birth (7,8),

斤om poor quality sources, thereby producing potable water

cryptosporidiosis in pigs has been reported to be delayeduntilthat meets the accepted drinking water standards, there is still

alter weanlng. CTWtOSPOridium infection, at the same time,

serious concem that the barrier to microbial breakthroughin

has been detected in plgS ranglng丘om 1 to 30 Weeks old by

the finished water may fail to measure up to our expecta‑

histological examinations in Cana血(9).

tions･ Any momentary or intemittent break(S) in the water

In the present study, the prevalence of CTyPtOSPOridium

treatment chain could allow substantial levels of pathogenlC

infection in asymptomatic, 1‑3 months old weaned piglets

microbes to enter potable wateトthe丘nal product. The recent

and 6 months old fattening porkers, the manure of which haslarge‑scale outbreaks of Cywtospon'dium in many industrial‑

been recognized to be one of the major sources Of contaminat‑

ized countries have demonstrated that conventional drinking

lng Surface water in Japan, was investigated in the central partwater treatment may not always be adequate to prevent water‑of Japan.

bome disease transmission (I). To date, there have been twowaterborne outbreaks due to CTyPtOSPOridium infection
reported in Japan. The first took place in Hiratsuka clty ln
August and September of 1994 and consisted of461 diarrhea

Fecal specimens: Fecal samples were obtained at thecases (2)･ The other occurred at a small town in Saita甲aslaughterhouse from 232 weaned piglets ( I and 3 months old)

Prefecture approximately 50 km northwest from Tokyo, lnand 252 fattening porkers (6 months old) in 8 stock‑raisingearly June and mid‑July of I 996and consisted of 8,8 I 2 patientsfarms located in Kanagawa Prefecture, Japan,from June I 998
to June 2000 (Table 1 ). One of the stock‑raising fans (FarmA) is located near a tributary of the Sagafniriver system･ Pigs

MATERIALS AND METHODS

who met the case definition (3). Theunusual resistance of00cysts such as Cmptospon'dium to chlorine‑based disinfTectants

were assigned to one of three age categones: 1, 3 and 6 months(4) allows for penetration of viable oocysts into the treatedwater supply.

according to information provided by the famers. Intestinal

Since the first report of cryptosporidial infections in plgS

(fecal) samples were collected either from the area between

in 1 977 (5,6), porcine cryptosporidiosis has been reported in

the cecumto the upper part of the colon of the piglets or kom
the rectum of the porkers at sacrifice. Cryptosporidial oocysts*Corresponding author: Tel: +8 I‑3‑5285‑1 1 1 1 , Fax: +81‑3‑5285‑

were detected in fecal samples from both the cecum/colon

1 173, E‑mail: tendo@nih.gojp

and the rectum ofinfectedanimals. Fecal samples were stored23

Table 1. A list of samples examined in the present study
ramGroup(age)No.

sampled148

AB

2 44 8ト3 months oldC
D
Subtota1

232

AE
F

∠U 2 76 months oldG1

HSubtota1

2Tota152484

Fig. 1. Differentialinterference contrast image ofoocysts of Cryptosporidium
ParVum･in a refrigerator until examination.

Cryptosporidial oocysts obtained &omfresh feces were fully sporulatedwith four sporozoites, either spherical or ovoid, with diameters ofAnalysis of C.LlarVum OOCyStS: Detection of oocysts was

4.5‑5.4 × 4.215.0 LLm.

performed using the formalin ethyl acetate (FEA) centrifugal
concentration method, followed by immunofluorescent stain‑
ing. Briefly, the intestinal content (fecal) Sample was diluted
twice with water, mixed thoroughly, andfi1tered with double‑

calculated using twoISided 95% confidence intervals (CI)･sheeted gauze to remove large debris. The filtrate ( ≦ 2 ml)

Differences in proportionsobserved were compared by Fisher 's

was diluted to 10 ml with water, supplemented with 3 ml ofexact test or x2‑test,and probability values less than 0.05 were

ethyl acetateand mixed thoroughly by shaking vlgOrOuSly in aconsidered significant.

screwICaPPed centrifuge tube. It was immediately centrifuged
at 3,000 rpm for 10 min. The sediment was collected, resus‑

RESUI.TS

pended with a small amount of water (≦ 100 JLl), smeared
onto a slide glass and血ied. The smear was then丘xed with

The study was undertaken to detemine the prevalence ofmethanoland stainedusing a direct immunofluorescent staining

C. payvum oocysts infection in plgS With respect to the age ofkit, FITC‑Conjugated mouse monoclonal antibody specificthe animal, locations (farms) and month of fecal collection.

for C･ panmm oo.cyst (MELIFLUOR㊥, Meridian, Ohio, USA)･

Fresh fecal samples were collected from 232 weaned piglets

The stained speclmen Was examined uslngfluorescent microI(1 and 3 months old), most of which were specially produced

scopy with the excitation wavelength at 450‑490 nM･for Chinese restaurants, and from 252 porkers at 6 months of

Statistical analysis: In the present study we examinedage, the body weights of which were approximately 100 kg.

demographic and environmental factors potentially associatedwith the excretion of C. parvum oocysts in the feces of 1‑6

The oocysts detected by immunofluorescent stainlng Wereconfirmed to be C. parvum by their morphologlCal features,months old pigs. These included age of the animal, geographic

including the size, shape, and intemal stmcture of oocysts,location (farrhs) and month of fecal collection. The distribution

and the thickness of the oocyst wall, as observed underof oocyst infection in plgS Was Calculated for each variable;

differential interference optics (Fig. 1 ).
Infection with C. payvum oocysts was associated with age

the associated odds ratios f♭r levels within each variable wereTable 2. Prevalence and crude odds ratio for excretion of Cryptosporidium pafVum
oocysts in porkers

Factor Prevale.n.cceyso.:Eo).r"um Cru(d,e5諾dcsI;atio pvalueram A

WeanedBCD

piglets

52/148 (35.1%)

2.7 (0,6‑12.8)

ns**

10/24 (41.7%)

3.6 (0.6‑20.0)

ns

13/48 (27.1%)

1.9(0.4‑9.6)

ns

2/12 (16.7%)

I.0*

Age (month)weaned1

69/213 (32.3%)

89.6 (12.3‑652.9)<0.0013

8/19 (42.1%)

136 (15.61‑186.4)<0.0016(FamA)

1/187 ( 0.5%)

I.0*

1‑3

77/232 (33.2%)

125.2(17.2‑909.2)

6

1/252

1.0*

piglets

Fatteningporkers

Wean edpiglets
Fatten lng(0.4%)

porkers* Referent category for the odds of excreting C. parvum oocysts.

** Not significant.

24<0.001

Table 3. Point prevalence and crude odds ratio for excretion of

Porcine cryptosporidiosis, first reported in 1 977 (5,6), has

Cryptospon'dium parvum oocysts according to month of fecal

since been found in many countries around the world. Pigs

collection in weaned piglets on ram A

Monthof sample Prevalence ofacquisition C. payvum oocysts
1999

Ju1

8/12 (66.7%)

Sept

1/8

Oct

2000

1.0

*were found to excrete oocysts in their feces (10). In other

4.7 (1.0, 21.7)(12.5%)

0.3

5/8 (62.5%)(0.03,3.3)

4/8 (50.0%)

2.3 (0.4, 12.6)

Dec

9/12 (75.0%)

7.0 (1.4, 35.3)

2/8 (25.0%)

0.8 (0.I, 5.0)

Feb

I/12Mar

0/20

Apr(8.3%)(<0.05studies, C. parvum infection has been reported to occur inns**

plgS Of all age groups, but inruminants only before weanlng∩ ∩ o.∩ ∩ ∩ nsn n0 s s s

3.9 (0.7, 21.7)

NovJan0.20%)

9/20 (45.0%)

CfフPtOSPOridium oocysts in Canada (9). Based on examina‑tion ofrectal swabs, 1 0 of 200 (5%) market pigs in Califbmia

6/20(30.0%)Augranglng from 1 to 30 weeks have been shown to excrete

Crudeodds ratio Pvalue(0.02,(7,8)･ ln the present study, 1 ‑3 months old piglets were shown

to excrete C. parvum oocysts in their feces. On FarmA,<2excretion or oocysts was detected in an overall average of

35.1% of animals, ranglng aom O% in March of2000 to 75%
in December of 1999. The odds of C. paTVum infection were1.9)0.0

slgnificantly greater in ll3 months old piglets than in porkers

1.9 (0.5, 7.0)of 6 months old, whereas there was no slgnificant differencein odds of infection between 1 month and 3 months old piglets.

May

5/12 (41.7%)

1.7 (0.4, 7.4)

Jun

2/8 (25.0%)

0.8 (0.1, 5.0)

Other epidemiologlCal surveys have also reported that infec‑* Referent category forthe odds of excreting C. parvum oocysts.
** Not significant.

tion is prevalent in recently weaned piglets and at the firstof pigs but not geographic location or month offecal collection.

stages of fattening; the rates were 32.6% in Nigeria (ll),19.9% in Korea (12), 41.5% in Trinidad and Tobago (13) and
59.2% in Spain (14). On the other hand, management offarmoperations has been shown to influence the course ofThe overall prevalence of infection with C. parvum oocysts

cryptosporidial infection in both nurslng and weaned piglets

among weaned piglets from four different farms was 33.2%(77/232), ranging from 16.7% (2/12) to 41.7% (10/24). Point(15).
In summary, the results obtained in the present study

prevalences of infection among 1 month and 3 months olddemonstrate that CりptOSPOridium infection is prevalent inweaned piglets were 32.3% (69/213) and 42.1% (8/19),

weaned piglets orト3 months old in Japan, supportlng thewhereas that among 6 months old fattenlng POrkers was 0.3%

idea that cryptosporidiosis is prevalent among young farm(1/252), ranging from O% to 0.5% (1/187) (Table 2). The age

animals. It is thus not unreasonable to assume that weanedof animals was slgnificantly associated with the odds ofpiglets, together with other young farm‑animals, are themajor reservoirs and transmission sources of this microbe,

infection by C. paTVum OOCyStS. The odds of infection among
1 and 3 months old piglets were approximately 90 to 136 times

a conclusion with major epidemiologlCal implications fわrgreater than that among 6 months old porkers from FarmA,

humans due to potential contamination of drinking water. The

where 95% CI were 12.3‑652.9 and 15ふ186.4, respectively

was 0.6 (95% CI: 0.3 to 1.7), suggesting that the categories orfinding that Cryptosporidium oocysts have contaminateddrinking water sources suggests that there is hydrologlC COmmeC‑
tion between livestock manure and such water bodies. And in

I and 3 months of age could be combined into one category.

fact, our recent epidemiologlCal finding that a larger number

Although there were slight differences in polnt Of prevalenceof fecal excretion of C. parvum among weaned piglets on
different farms (Farms A‑D), the odds ratio of excretion ofthrough catchment areas where many hoggeries are located(Table 2). The odds ratio calculated between 1 and 3 monthsof Cywtosporidium oocysts occurred in tributariesrunnlng
might lend circumstantial evidence to support this idea ( 1 6).

oocysts among piglets on Farm B was 3.6 (95% CI: 0.6 to20.0) times greater than that among piglets on ram D.

ACKNOWLEDGMENTS

To evaluate the oocyst excretion in feces with respect toweaned piglets on one of the raising farms (Farm A) was

We thank Dr. Yukiya Fukuma, Dr. Yuichiro Nozawa, andthe staffs of the Meat Inspection Stations in Hiratsuka andmeasured monthly from July 1999 to June 2000 (Table 3).

Atsugi, Kanagawa, for providing fecal specimens during theThe polnt Prevalence of oocyst infection fluctuated according

study.

to the month of fecal collection, ranglng from O% in Marchof2000 to 75.0% in December of 1999. The odds ofC.pafWm

This study was partly supported by a Health Science
Research Grant on Emerglng and Re‑emerglng Infectious

infection among piglets tested in August and December ofDiseases (12103 101) from the Ministry of Health, Labourand Welfare, Japan.

month of fecal collection, the prevalence of infection among

1 999 were 4･7 and 7.0 times greater, respectively, than among

piglets tested in June 1999.

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26