The seroepidemiology of toxoplasmosis in the lower Fraser Valley of British Columbia

OBJECTIVE: To detem1ine the seroprevalence of toxoplasmosis in vegetarian and non vegetarian members of different elhnic communities in Lhe lower Fraser Valley of Bt;tish Columbia . DESIGN: Serum samples were collected from 2027 participants drawn from various ethnic groups and tested by ELISA for U1e presence of immunoglobulin (lg) G and IgM antibodies to Toxoplasma gondii. Coded questionnaires requesting infom1ation relevant to the study were completed by each participant. The study population comprised 1334 females and 693 males: ages ranged from 17 lo 102 years . MAIN REsULTS: Four hundred and nineteen (20.7%) indiv;duals were IgG positive with lilres ranging from I: 100 lo I :3200. IgM antibodies were detected in only four indiv;duals. The seroprevalence rose wilh increase in age but U1ere was no signi ficant difference between males and females. A positive correlation was shown between ingestion of meat and between consumption of unpasteurized milk and antibodies lo T gondii. Eighty per cent of females between the ages of 17 and 40. of all eU1nic origins. were seronegative. Seropositiv;Ly did nol differ between cal owners and non-cal owners. CoNCLUSIONS: Women of childbearing age are at risk of acquiring toxoplasmosis during pregnancy and of transmitting the infection lransplacenlally. Consumption of undercooked meal and unpasteurized milk may result in Lhe acqu isition of toxoplasmosis. Data suggest Lhal acquisition of toxoplasmosis is more likely via env;ronmenlal oocysls or cysts in food source animals than by direct contact with cats.


Seroepidemiology of toxoplasmosis
PRlNCIPAUX REsULTATS : Qualre cenl dix-neuf (20. 7 o/o) sujels elaienl seroposiUfs a r egard de l'I gG avec des lilres variant de I : I 00 a I :3 200.Des anlicorps anli-lgM onl ele deceles chez qua(J•e individus seu lemenl.La seroprevalence s'elevail avec rage.mais on n'a observe aucune d ifference significaUve enu•e les hommes elles femmes .Une correlalion posilive a ele elablie enu•e ingeslion de v iande el consommation de !ail non pas leu rise el Ia presence d'anlicorps an li-T gondii.Qualre-vingl pour-cenl des femmes agees de 17 a 40 ans.de loules origines elhniques.elaienl seronegalives .La seroposilivile n•a pas differe.que les sujels soienl ou non proprielaires de chats.CoNCLUSIONS: Les femmes en age de procreer sonl exposees a un risque de loxoplasmose duranl la grossesse qui peul se lransmellre au foelus.La consommaUon de viande insuffisammenl cuile ou de !ail non pasleUiise pourrail cnlrainer une loxoplasmose.Selon les donnees.l'acq uisiUon de Ia loxoplasmose est plus susceplible de se produire par conlaminalion d'Oiigine environnementale provenanl de sources alimenlaires animales que par contact direct avec les chats . O VER TilE PAST 30 YEARS TIIERE I-LAS BEEN A STEADY growth in the literature of articles on toxoplasmosis.indicating an increasing awareness on the part of physicians and veterinarians.The causative organism, Toxoplasma gondii, is a protozoan parasite which was first discovered in a North African rodent in the early part of the century (l).T gondii is ubiquitous in nature, and over 300 species of mammals and 30 species of birds have been identified as intermediate hosts of lhis parasite (2).
Humans acquire the infection either by eating undercooked meal containing viable cysts or by accidental ingestion of oocysls shed by infected cals.The relative importance of these lwo rou les of infection is not clear.Nevertheless, the majority of infections is subclinical and passes unnoticed.Infection may also be acquired congenitally, as a result of organ transplantation or through lhe ingestion of unpasteurized milk, in particular goal's milk (3,4).Infection with T gondii is common throughout the world and stud ies have shown lhal 25 to 60% of lhe adulls in bolh Canada and lhe United Stales have been exposed lo this organism at some time in their life (5)(6)(7)(8)(9).
The aim of lhis study was lo determine lhe seroprevalence of toxoplasmosis in vegetarian and nonvegelarian members of different ethnic communities in British Columbia.Those individuals who are at highest risk of acquiring toxoplasmosis are identified, as well as some of the factors that may predispose lo infection wilh this parasite.The serological findings are presented and analyzed with reference lo lhe demographic dala.

PATIE NTS AND METHODS
Seven millilitres of blood were drawn from each of 2027 participants, a ll of whom signed a consent form and completed a coded questionnaire.The participants were drawn from members of the East.Indian Sikh, Moslem and Hindu communities.the Native community, Chinese, Japanese and other Asian communities.and from Caucasians of various ethnic origins .Participants who did not speak or read English were assisted with the completion of the questionnaire by an interpreter.
Specimens from lhe various East.Indian communities were obtained during visits lo lhe respective lem-pies and mosques at.U1e time of their Sunday worship.Participation by members of the Native community was solicited through the Down Town Vancouver East Hastings Community Centre and a health unit.Other participants were sought.from among U1e staff members of various laboratories, hospitals and institutions, including homes for the elderly, in the lower Fraser Valley region of British Columbia .
The questionnaire requested information on age, sex, dietary preferences, type of meal consumed and to what degree the meat was cooked (rare, medium rare elc).consumption of unpasteurized milk, occupation, cat ownership and, in the case of females, whether the individual was pregnant.at. the time of completion of the questionnaire.
Serum was separated immediately, coded to correspond lo lhe questionnaire and stored at -20°C .Specimens were tested for lhe presence of antibod ies lo T gondii by the Toxonostika IgG and IgM (Organon Technika) ELISA .
Statistical analysis was performed using Student's L lest.x 2 test.and analysis of variance.

RESULTS
The 2027 individuals of various ethnic origins who provided blood specimens and completed questionnaires ranged in age from 17 to 102 years.with a median age of 55 and a mean age of 39.8.Sixty-six per cent. of lhe study population was female (mean age 38.6 years) and 34% was male (mean age 42.2), wilh no significant.difference between male and female (P>0.05).
Four hundred and nineteen of lhe 2027 (20.7%) individuals tested by ELISA immunoglobulin (!g) G had antibodies lo T gondii, with titres ranging from 1: 100 to 1:3200 (Figure 1).Only four individuals were positive by ELISA IgM: three with titres of 1: 100 and one with a titre of 1:400.There was no significant difference in seropositivity between males (22.4%) and females (19.5%) (P>0.05).
The Caucasian participants (1112) numbered more lhan half of the study population and 225 (20.2%) were seropositive.Forty-five per cent (915) of all the participants were non-Caucasian and 194 (21.2%) were seropositive.The East Indian participants comprised 677 of   cant difference between male and female seropositvily in the different age groups (P>0.05).
Females comprised 66% (1338) of the study population.Of this number 261 (19.5%) were ELISA IgG positive.Titres of 1:800 or greater were demonstra ted in a fifth of those who were seropositive.However, none of the individuals with high levels of IgG antibodies were IgM positive.
Forty-nine of the female participants were pregnant, all except three were nonvegetarians and only 10 had serological evidence of exposure to toxoplasmosis.The seropositive females all consumed meat, seven owned a cat and four regularly drank unpasteurized milk.Twothirds of the pregnant females were seronegative and 75% of them consumed meat and owned a cat.. Two of three who were vegetarians and five nonvegetarians also drank unpasteurized milk.Eighty-four per cent (246) and 82% (459) , respectively, of non-Caucasian and Caucasian female participants within the childbearing years of 17 to 40 were seronegative (Figure 3).Dietary preferences: Of 260 (13%) vegetarian participants, 107 (41 %) had consumed meat at some stage of their life.The strict vegetarians, therefore , totalled 153.The majority of vegetarians were East Indians, with only 22 of260 (8.5%) being Caucasians.Forty-one non-Cau-casians and four Caucasian vegetarians were ELISA IgG positive.
Twenly-lwo per cent of non-Caucasians and 20% of Caucasians who had consumed meal, in one form or another, were seropositive (Figure 4).This was shown lo be slalislically significant (P<0.05).Table 1 shows lhe breakdown by elhnicily and antibody slalus in relation lo lhe degree lo which consumed meal was cooked.
Analysis of lhe different types of meal consumed found a seroprevalence among non-Caucasian participants ranging from 17.2% for pork consumption lo 25.2% for those who consumed goal meal, with an a verage of 21.2% overall.By contrast.lhe seroprevalence among Caucasians ranged from 18.5% for poultry consumption to 19.8% for pork.wilh an average of 19.4% for six types of meal.Among Orientals, 24 of 159 (15%) and 23 of 160 (14%) who consumed mainly poultry and beef were seropositive .Of the 425 seronegative non -Caucasians, 234 (55%) were East Indians who regularly consumed beef.Risk factors -Cat ownership: Ninety-four of 488 (19.2%) individuals who had owned a cal at some stage of their lives were ELISA IgG positive.This was nol significanlly different from lhe non-cat owners (P>0.05).Eighty-seven (93%) of lhe seropositive individuals were Caucasian and 72 of this group were between 26 and 52 years of age.By conlrasl55 (11.3%) and 339 (69 .5%)non-Caucasian and Caucasian cal owners.respectively.were seronegative (Figure 4) .An1ong Orientals , one of 21 cal owners and 21 of 141 non-cal owners were ELISA IgG positive.Risk factors -Occ u patio n associa t e d with cat s and m eat h andling : Of the 202 participants who indicated lhal their occupation fell inlo either of these lwo categories, 51 were seropositive (Figure 4).This was statistically s ignifican l (P<O.05).The serological findings of one individual-ELISA !gG greater than 1:3200, ELISA lgM 1:400 and immunofluorescent assay-IgG 1:2048-were diagnostic of acute toxoplasmosis.This participant was a meat-cutter who had recently developed 'flu -li ke symptoms, al which time a lymph node was biopsied.He also owned a cal.

Ris k fact ors -Co n sumption of unpasteurized milk:
There was a significant difference in seropositivity between those who consumed unpasteurized milk and those who consumed pasteurized milk (P<0.05) .Fiftyfour per cent of the participants, 4 72 Caucasians and 616 non-Caucasians, indicated that they had at some stage consumed unpasteurized m ilk (Figure 4).Twentytwo per cent and 23%, respectively, of Caucasians and non-Caucasians within th is grou p were seropositive.By contrast, of the 939 individuals who h ad never consumed unpasteurized milk.116 (12%) Caucasians and 51 (5%) non-Caucasians were ELISA IgG positive .Seven of30 (23%) Orientals who had consumed unpasteurized milk were seropositive.

DISCUSSION
The age-specific seropositivity in the present study was considerably lower than that.reported by other investigators.who have shown that the seroprevalence in popu lations studied in southern Ontario and Holland climbs lo approximately 45% between the ages of 21 and 25 years and reaches a maximal value of 59% in the age groups between 40 and 79 (10,11) .Although prevalence rates vary throughout the world and are influenced by geographic.climatic, ethnic and socioeconomic factors , in most populations tested there appears to be an increase with age, suggesting continued exposure throughout life (12)(13)(14) .The seroprevalence recorded in the age groups above 50 years in the present study is not comparable to other findings because the number of participants was too small.
The study has shown that, in both the non-Caucasian and Caucasian communities, in the childbearing age group in this region of British Columbia.approximately four of five females are nonimmune and therefore at risk of acquiring toxoplasmosis during pregnancy and of transmilting the infection to the fetus.By contrast., one in four seropositive females over the age of 40 years. of all ethnic origins, had antibody levels of 1:800 or greater by ELISA.a levellhat is considered to be indicative of a more recent infection.However, IgM antibodies associated with acute infection were not demonstrated in any of the female participants with high levels of IgG.
Members of the different religious groups within the East Indian community were included in the study becau se of differ ences in th e type of food consumed.particularly meat.as dictated by th eir religious beliefs.The Hindu and Moslem diets exclude beef and pork.respectively, whereas the diet of Sikhs and most Caucasians is indiscriminate.While the majority of vegetarians was East Ind ian. the 43% who had consumed meal had done so for only brief periods of lime, usually during the early days of immigration lo Canada.Although the consumption of raw or undercooked meal is regarded as one of the major routes of acquisition of toxoplasmosis, only 3% and 17%, respectively. of the seropositive individu a ls consumed rare or medium rare meal.By comparison rare or medium rare meal was consumed by 22% of the ELISA-n egative individuals .The percentage of seropositive Caucasians who consumed rare lo medium rare meal was U1ree limes greater than non-Caucasian seroposilives, a difference lhal is not too surprising given that the non-Caucasians comprised mainly East Indians who, in the preparation of their traditional dishes, cook their meal extremely well .
The consumption of lamb and pork has been shown to pose a greater risk of acqu isition of toxoplasmosis than consumption of other types of meal (15)(16)(17)(18)(19). yet 76% and 80% of non-Caucasians and Caucasians.respectively.who consumed pork were seronegative.Similarly.the Orientals in the study indicated a preference for poultry.another possible source of infection (20): yet only 15% were seropositive.suggesting lhallhe method of cooking may prevent the acquisition of toxoplasmosis.
The difference of 19.2% and 20.8% in seroprevalence between cal owners and non-cal owners, respectively.was not statistically significant.This finding is in marked contrast lo the findings in a Seallle.Washington study.in which the seroprevalence in cal owners was considerably greater than among those who denied ever having a cal as a pel (21).
In the present study cal ownership.either present or past.was much more prevalent among Caucasians than non-Caucasians and among females than males .Many members of the East Indian community expressed a distinct dislike of cats and preferred lo have no contact with the animals.
Numerous studies and several family and community or group outbreaks have pointed lo U1e major role played by cats in the transmission of toxoplasmosis.both through direct contact wilh humans and by contamination of Ule soil with oocysls (22)(23)(24)(25)(26) .The seroprevalence in Ule present study of 28% and 24% in non-Caucasians and Caucasians.respectively.who had never owned or had direct contact wiUl cats.points lo oocysls in the environment and/or tissue cysts in consumed meal as the more likely source of infection in this group.
T gondii-induced reproductive losses in goals have been reported from most countries around the world (27)(28)(29)(30)(31). and unpasteurized goal milk has been identified as the source of infection in al least two outbreaks (3,4).While none of the participants acknowledged drinking raw goal's milk.more than 50% of U1e individuals admitted lo consumption of unpasteurized milk whenever the opportunity presented itself.The East Indian participants, who constituted 33% of the study population , indicated that it was a regular practice to drink unpasteurized buffalo milk.both before their immig ration lo Canada and on return visits to India.Many of the Indo-Canadian participants commented on their preference for unpasteurized milk.irrespective of the source.While the sale of raw milk is illegal in British Columbia.il is evid ently readily available and frequently purchased by members of the East Indian comm unity.
Although lillie is knO\:vn about toxoplasmosis in water buffalo, the consumption of milk from this anima l may account for the presence of antibodies in the 23% of non-Caucasians found lo be positive by ELISA IgG.Studies have shown that the prevalence of antibod ies lo T gondii in buffalo sera in India range from l 0 lo 24% (32)(33)(34).
In the present study a litre of 1: l 00, the first dilution recorded , was generally regarded as a borderline result.or the cut-off poinl.In generaL titres of 1:800 to 1: 1600 or greater together with a positive lgM reaction are considered to be indi cative of recent acute infection.However.studies have shO\vn that the presence of IgM-specific antibody may not be the ideal indicator of recent infection.s ince the persistence of these antibodies varies greatly from individual lo individual (35).
Two of the three participants with positive lgM reactions of 1:100 were Caucasians.Both participants had at some stage owned a cal.were nonvegelarians and had on occasion consumed unpasteurized milk.The third participant with a similar litre was a nonvegelarian Hindu male who did not ovm a cal but had regularly consumed unpasteurized milk.The participant with the IgM litre of 1:400 was a Caucasian male.who owned a cal but did not drink unpasteurized milk.He was a meal culler by profession, and several weeks previously in the course of his work had cut his hand.Histological findings of a lymph node biopsy, taken al about the same lime as the serum sample for this study.were considered lo be consistent with toxoplasmosis.suggesting that.the infection had been acqu ired from meal he was handling at. the lime or from the meat block.In this instance.the high IgG litre and the positive IgM were considered to be diagnostic of acute toxoplasmosis.The low IgM litre of the other three participants is suggestive of an infection acquired at some earlier date.
In conclusion, the seroprevalence of the population studied was found lobe 20.7%, a finding lhal is considerably lower than the 40% recorded for British Columbia in 1976 (36) or the 28% determined by meU1ylene blue dye lest in inhabitants of the Greater Victoria area in 1977 (37).The previous finding of 40% was based on serum samples from all age groups from across the province submitted over a period of 16 years for routine lesling for toxoplasmosis, and very likely reflects seasonal variations and the anticipated higher incidence associated with individuals from rural parts of the province.On the other hand, the present finding is in all probability a relatively accurate estimate of the overall seroprevalence of toxoplasmosis in the mainly urban and suburban area of the lower Fraser Valley of British Columbia .While the cat is known to be central to the dissemination and acquisition of toxoplasmosis, based on the present epidemiological findings, it appears that indirect transmission by way of oocysts in the environment and hence via cysts in food source animals is a more likely method of acquiring toxoplas-

Seroepidemiology of toxoplasmosis
mosis than by direct contact with cats.The possible role of unpasteurized milk.and particularly buffalo milk, in the acquisition of toxoplasmosis is an interesting one and should be further investigated.The present study h as also shown that over 80% of females of a ll ethn ic origins in the childbearing age group are seronegative and therefore at risk of acqu iring toxoplasmosis during pregnancy and of transmitting the infection transplacentally.

Figure 4 )
Figure 4) Toxoplasmosis risk factors versus ELISA immunoglobulin G results; n=2027 CAN j INFECT DIS VOL 5 No 5 SEPTEMBER/OCTOBER 1994 Seroepidemiology of toxoplasm osis

TABLE 1 A
ntibody sta tus of ethnic groups by degree of cooking of meat c onsumed