1747 Journal o f Food Protection, Vol. 77, No. 10, 2014, Pages 1747-1753 doi: 10.4315/0362-028X.JFP-14-167
Detection and Survival of Toxoplasma gondii in Milk and Cheese from Experimentally Infected Goatsf J. P. DUBEY,1* S. K. VERMA,1 L. R. FERREIRA,1S. OLIVEIRA,1 A. B. CASSINELLI,' Y. YING,1 O. C. H. KWOK,1 W. TUO,1 O. A. CHIESA,2 and J. L. JONES3 'U.S. Department o f Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350; 2Division o f Applied Veterinary Research, Office o f Research, HFV-520, Center fo r Veterinary Medicine, U.S. Food and Drug Administration, MOD II - 8401 Muirkirk Road, Laurel, Maryland 20708; and 3Division o f Parasitic Diseases and Malaria, Center fo r Global Health, Centers fo r Disease Control and Prevention, 1600 Clifton Road, Mailstop A-06, Atlanta, Georgia 30333, USA MS 14-167: Received 11 April 2014/Accepted 30 May 2014
ABSTRACT The consumption of unpasteurized goat cheese and goat’s milk has been suggested as a risk factor for toxoplasmosis in humans. In the present study, detection and survival of Toxoplasma gondii in milk and cheese was studied by bioassay in mice (milk) and in cats (cheese). Eight goats were inoculated orally with 300 to 10,000 oocysts of T. gondii strain TgGoatUS26. Milk samples were collected daily up to 30 days postinoculation and bioassayed in mice and cats. For mouse bioassay, 50 ml of milk samples were centrifuged, and the sediment was inoculated subcutaneously into mice. Mice were tested for T. gondii infection by seroconversion and by the demonstration of parasites. By mouse bioassay, T. gondii was detected in milk from all eight goats. The T. gondii excretion in milk was intermittent. For cat bioassay, 400 ml (100 ml or more from each goat) of milk from four goats from 6 to 27 days postinoculation were pooled daily, and cheese was made using rennin. Ten grams of cheese was fed daily to four cats, and cat feces were examined for oocyst shedding. One cat fed cheese shed oocysts 7 to 11 days after consuming cheese. Attempts were made to detect T. gondii DNA in milk of four goats; T. gondii was detected by PCR more consistently, but there was no correlation between detection of viable T. gondii by bioassay in mice and T. gondii DNA by PCR. Results indicate that T. gondii can be excreted in goat’s milk and can survive in fresh cheese made by cold-enzyme treatment. To prevent transmission to humans or animals, milk should not be consumed raw. Raw fresh goat cheese made by cold-enzyme treatment of unpasteurized milk also should not be consumed.
Toxoplasma gondii infects virtually all warm-blooded animals, including humans, livestock, and marine mammals (14). In the United States, various surveys have found that 10 to 50% of the adult population has been exposed to this parasite (20). T. gondii infection can cause mental retardation, loss of vision, and other congenital health problems in human infants. Humans usually become infected postnatally by ingesting tissue cysts in undercooked meat and by consuming food or drink contaminated with oocysts. Toxoplasmosis continues to be a public health problem in the United States. Annually, approximately 1 million people are infected with T. gondii, and over 4,800 people develop symptomatic ocular disease (30). A recent study identified drinking unpasteurized goat’s milk as a risk factor for recently acquired toxoplasmosis in pregnant women in the United States (29). Feeding of goat whey was also identified as a source of T. gondii infection in pigs in The Netherlands * Author for correspondence. Tel: 301-504-8128; Fax: 301-504-9222; E-mail: [email protected]. t The findings and conclusions in this report are those of the authors) and do not necessarily represent the views of the Department of Health and Human Services or the Centers for Disease Control and Prevention or the U.S. Department of Agriculture.
(33). Clinical and even fatal toxoplasmosis has been reported in humans after drinking unpasteurized goat’s milk (35, 37, 42). Pasteurization has been demonstrated to kill T. gondii (16). Unpasteurized, raw milk is sold by small-operation goat farmers, and goat cheeses made from raw milk could be a source of T. gondii infection. It has been more than 60 years since T. gondii was demonstrated in milk of mice (24). Since then, T. gondii was found in milk of many other hosts. However, interest in transmission of T. gondii via milk declined when the full life cycle was discovered in 1970 (14) and pasteurization of milk became mandatory for commercially sold milk; pasteurization kills T. gondii. Recently, there has been an upsurge in organic farming and the consumption of raw milk or milk products made from unpasteurized milk. There is great concern regarding whether it is safe to consume raw (unpasteurized) milk (http://www.cdc.gov/foodsafety/raw milk/raw-milk-index.html). Many Americans consume products labeled as “ only for pet consumption” or as participants in food cooperatives (21). Goat’s milk is consumed by children who are allergic to cow milk because it is easily digested and readily available. The present investigation was prompted because drinking unpasteurized goat’s milk was found to be a risk factor for recently
J. Food Prot., Vol. 77, No. 10
DUBEY ET AL.
acquired toxoplasmosis in pregnant women in the United States (29). The objectives of the present study were to determine frequency of excretion of viable T. gondii in milk of experimentally infected goats and to test if the parasite remains viable in cheesemaking procedures.
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Serological examination. Sera of goats were tested for T. gondii antibodies with the modified agglutination test (MAT), as described by Dubey and Desmonts (17). Sera were diluted twofold with 1:25 to 1:3,200. A titer of 1:25 was considered evidence of T. gondii exposure. Of the eight goats selected for the present study, seven did not have T. gondii antibodies at 1:25 dilution; goat F was positive at 1:50 but was used in the experiment because of the borderline result.
s; .1
Cheesemaking and survival of T. gondii in cheese. To study the survival of T. gondii during the cheesemaking process, whole unpasteurized milk from an uninfected goat and cell-culturederived tachyzoites of the GT1 strain of T. gondii (5) were used; the GT1 strain is highly virulent for mice; thus; bioassay results are obtained earlier than with other strains. This strain was used only for standardization of techniques and not to infect goats.
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Detection and Survival of Toxoplasma gondii in Milk and Cheese from Experimentally Infected Goatsf J. P. DUBEY,1* S. K. VERMA,1 L. R. FERREIRA,1S. OLIVEIRA,1 A. B. CASSINELLI,' Y. YING,1 O. C. H. KWOK,1 W. TUO,1 O. A. CHIESA,2 and J. L. JONES3 'U.S. Department o f Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350; 2Division o f Applied Veterinary Research, Office o f Research, HFV-520, Center fo r Veterinary Medicine, U.S. Food and Drug Administration, MOD II - 8401 Muirkirk Road, Laurel, Maryland 20708; and 3Division o f Parasitic Diseases and Malaria, Center fo r Global Health, Centers fo r Disease Control and Prevention, 1600 Clifton Road, Mailstop A-06, Atlanta, Georgia 30333, USA MS 14-167: Received 11 April 2014/Accepted 30 May 2014
ABSTRACT The consumption of unpasteurized goat cheese and goat’s milk has been suggested as a risk factor for toxoplasmosis in humans. In the present study, detection and survival of Toxoplasma gondii in milk and cheese was studied by bioassay in mice (milk) and in cats (cheese). Eight goats were inoculated orally with 300 to 10,000 oocysts of T. gondii strain TgGoatUS26. Milk samples were collected daily up to 30 days postinoculation and bioassayed in mice and cats. For mouse bioassay, 50 ml of milk samples were centrifuged, and the sediment was inoculated subcutaneously into mice. Mice were tested for T. gondii infection by seroconversion and by the demonstration of parasites. By mouse bioassay, T. gondii was detected in milk from all eight goats. The T. gondii excretion in milk was intermittent. For cat bioassay, 400 ml (100 ml or more from each goat) of milk from four goats from 6 to 27 days postinoculation were pooled daily, and cheese was made using rennin. Ten grams of cheese was fed daily to four cats, and cat feces were examined for oocyst shedding. One cat fed cheese shed oocysts 7 to 11 days after consuming cheese. Attempts were made to detect T. gondii DNA in milk of four goats; T. gondii was detected by PCR more consistently, but there was no correlation between detection of viable T. gondii by bioassay in mice and T. gondii DNA by PCR. Results indicate that T. gondii can be excreted in goat’s milk and can survive in fresh cheese made by cold-enzyme treatment. To prevent transmission to humans or animals, milk should not be consumed raw. Raw fresh goat cheese made by cold-enzyme treatment of unpasteurized milk also should not be consumed.
Toxoplasma gondii infects virtually all warm-blooded animals, including humans, livestock, and marine mammals (14). In the United States, various surveys have found that 10 to 50% of the adult population has been exposed to this parasite (20). T. gondii infection can cause mental retardation, loss of vision, and other congenital health problems in human infants. Humans usually become infected postnatally by ingesting tissue cysts in undercooked meat and by consuming food or drink contaminated with oocysts. Toxoplasmosis continues to be a public health problem in the United States. Annually, approximately 1 million people are infected with T. gondii, and over 4,800 people develop symptomatic ocular disease (30). A recent study identified drinking unpasteurized goat’s milk as a risk factor for recently acquired toxoplasmosis in pregnant women in the United States (29). Feeding of goat whey was also identified as a source of T. gondii infection in pigs in The Netherlands * Author for correspondence. Tel: 301-504-8128; Fax: 301-504-9222; E-mail: [email protected]. t The findings and conclusions in this report are those of the authors) and do not necessarily represent the views of the Department of Health and Human Services or the Centers for Disease Control and Prevention or the U.S. Department of Agriculture.
(33). Clinical and even fatal toxoplasmosis has been reported in humans after drinking unpasteurized goat’s milk (35, 37, 42). Pasteurization has been demonstrated to kill T. gondii (16). Unpasteurized, raw milk is sold by small-operation goat farmers, and goat cheeses made from raw milk could be a source of T. gondii infection. It has been more than 60 years since T. gondii was demonstrated in milk of mice (24). Since then, T. gondii was found in milk of many other hosts. However, interest in transmission of T. gondii via milk declined when the full life cycle was discovered in 1970 (14) and pasteurization of milk became mandatory for commercially sold milk; pasteurization kills T. gondii. Recently, there has been an upsurge in organic farming and the consumption of raw milk or milk products made from unpasteurized milk. There is great concern regarding whether it is safe to consume raw (unpasteurized) milk (http://www.cdc.gov/foodsafety/raw milk/raw-milk-index.html). Many Americans consume products labeled as “ only for pet consumption” or as participants in food cooperatives (21). Goat’s milk is consumed by children who are allergic to cow milk because it is easily digested and readily available. The present investigation was prompted because drinking unpasteurized goat’s milk was found to be a risk factor for recently
J. Food Prot., Vol. 77, No. 10
DUBEY ET AL.
acquired toxoplasmosis in pregnant women in the United States (29). The objectives of the present study were to determine frequency of excretion of viable T. gondii in milk of experimentally infected goats and to test if the parasite remains viable in cheesemaking procedures.
o CO I V O CN (N
o 15
O CN CO
Serological examination. Sera of goats were tested for T. gondii antibodies with the modified agglutination test (MAT), as described by Dubey and Desmonts (17). Sera were diluted twofold with 1:25 to 1:3,200. A titer of 1:25 was considered evidence of T. gondii exposure. Of the eight goats selected for the present study, seven did not have T. gondii antibodies at 1:25 dilution; goat F was positive at 1:50 but was used in the experiment because of the borderline result.
s; .1
Cheesemaking and survival of T. gondii in cheese. To study the survival of T. gondii during the cheesemaking process, whole unpasteurized milk from an uninfected goat and cell-culturederived tachyzoites of the GT1 strain of T. gondii (5) were used; the GT1 strain is highly virulent for mice; thus; bioassay results are obtained earlier than with other strains. This strain was used only for standardization of techniques and not to infect goats.
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