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To the Editor: Toxoplasma gondii, a zoonotic protozoan parasite for which felids are the only definitive hosts, can infect humans and other warm-blooded animals. Transmission usually occurs orally from oocysts shed by felids in water and on food, through tissue cysts in undercooked meat, or transplacentally. In particular, young cats shed oocysts that can sporulate and become infectious within a day, depending on temperature and humidity. Sporulated oocysts can survive in moist soil for months to years (1). In September 2014, the number of dead squirrels reported to the Dutch Wildlife Health Centre and the Dutch Mammal Society increased suddenly. The red squirrel (Sciurus vulgaris) is the only species of squirrel endemic to the Netherlands. Members of the public claimed that squirrels were “dropping dead from trees.” Subsequently, the public was encouraged to report and submit dead squirrels. A total of 187 animals were reported through October 2014, of which 37 were submitted for necropsy. Necropsy included macroscopic examination; cytologic analysis of liver, spleen, lungs, and intestinal contents stained with hemacolor (Merck, Darmstadt, Germany); and histologic examination of samples of various organs fixed in formalin, embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin. For 8 adult animals, body condition (based on degree of fat storage and muscle development) was good; 12 juveniles were in poor condition. Typically, the trachea contained foam, and lungs were hyperemic and edematous. The liver was enlarged and pale, and the spleen was enlarged. In 13 animals, numerous small crescent-shaped organisms, with eccentrically placed nuclei consistent with tachyzoites of T. gondii, were identified by cytology in lung, liver, and spleen (2). Main histopathologic findings were pulmonary interstitial lymphoplasmocytic and neutrophilic infiltrates with edema and numerous intra-alveolar macrophages (17/20) and multifocal lymphoplasmocytic infiltrates with necrosis in the liver (13/20). Extensive splenic necrosis was occasionally observed (4/20). Intestines contained mild plasmacytic infiltrates. Numerous tachyzoites consistent with T. gondii were present in alveolar macrophages and epithelial cells, splenic macrophages, and hepatocytes. Duplicate slides were stained immunohistochemically by using polyclonal antibodies against T. gondii following a standard ABC protocol (3). Organisms stained for T. gondii in liver, spleen, lungs, and intestine. Toxoplasma was not detected in any brain. DNA was isolated (DNeasy Blood and Tissue Kit; QIAGEN, Hilden, Germany) from tissues of 14 squirrels and tested by quantitative PCR (1); T. gondii DNA was detected in 13. We successfully sequenced the T. gondii GRA6 gene for 11 squirrels and identified sequences to clonal type II T. gondii previously identified in sheep from the Netherlands (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU325790","term_id":"283580620","term_text":"GU325790"}}GU325790) (4). Incidental findings in the animals tested were encephalitis (2/20), coccidiosis (5/20), trauma (6/20), myocarditis (4/20), nephritis (1/20), lymphadenitis (1/20), and intestinal (3/20) and external (5/20) parasites. The remaining 17 animals showed >1 of the following pathologic conditions: hemorrhages consistent with trauma (12/17), mild to severe intestinal coccidiosis (12/17), pneumonia (3/17), splenitis (1/17), Taenia martis cysticerci (1/17), and external parasites (8/17). Immunohistochemistry results for all 17 were negative for T. gondii. On the basis of necropsy and molecular findings, we conclude that 20 of 37 examined squirrels died of disseminated T. gondii type II infection. These animals included adults and juveniles and were not restricted to specific geographic areas (Figure). The remaining animals died of trauma (12/17) or other causes (5/17). Figure Spatial distribution of wild red squirrels (Sciurus vulgaris) investigated for Toxoplasma gondii and classified by cause of death, the Netherlands, 2014. Red squirrels are susceptible to T. gondii, and infection can lead to death. However, in our sample, the proportion of squirrels that died of toxoplasmosis (>50%) was higher than in other studies (≈16%) (5–7). The apparent increase in squirrel deaths and unexpectedly high proportion of fatal T. gondii infections suggests a toxoplasmosis outbreak among red squirrels. Possible explanations for this surge in cases include increased exposure to the parasite, increased susceptibility to infection, or increased virulence of the pathogen. Clonal T. gondii type II, the strain most frequently involved in human cases and endemic to Europe and North America, was identified. An increased virulence of the pathogen could not be proven (8). On the basis of lymphoid hyperplasia in the spleen and lymph nodes, affected squirrels had no signs of immunosuppression. Thus, the most likely explanation is increased exposure to the parasite. Sources of infection for red squirrels are not known; however, oocysts shed in cat feces may contaminate the nuts, fungi, shoots, and berries that constitute the diet of the squirrel. Stray, unspayed cats are common in the Dutch countryside. More than 3 million domestic cats (Felis domesticus) exist in the Netherlands, including several tens of thousands of free-roaming cats that reproduce (9). Determining the exact source of infection is important because humans also harvest wild fruits, nuts, and fungi from these areas. This outbreak highlights that contamination of the environment with T. gondii oocysts is of concern not only from a public health viewpoint but from a biodiversity perspective as well (1,10).

Toxoplasma gondii is an important zoonotic parasite with a worldwide distribution. Felids are the definitive hosts and, upon primary infection, they shed millions of oocysts into the environment. These oocysts are infectious to a wide range of warm-blooded animals, and these intermediate hosts develop tissue cysts. Humans can be infected through oocysts, or by consumption of raw or undercooked meat containing tissue cysts. If a woman is infected for the first time during pregnancy, the parasite can be transmitted transplacentally, which may result in death of the fetus, or a baby born with nervous system abnormalities or chorioretinitis. Moreover, all people infected with T. gondii are at risk of developing ocular toxoplasmosis, and immunocompromised individuals are at risk for disseminated toxoplasmosis. Prevention strategies are usually aimed at pregnant women and are based on health education or early detection and treatment. Insight into the relative attribution of the different sources of infection and the possible intervention measures associated with these sources will aid the development of more effective prevention strategies that also will protect the general population.

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved.

Toxoplasma gondii is an important zoonotic pathogen that is best known as a cause of abortion or abnormalities in the newborn after primary infection during pregnancy. Our aim was to determine the prevalence of T. gondii in wild boar to investigate the possible role of their meat in human infection and to get an indication of the environmental contamination with T. gondii. The presence of anti-T. gondii antibodies was determined by in-house ELISA in 509 wild boar shot in 2002/2003 and 464 wild boar shot in 2007. Most of the boar originated from the "Roerstreek" (n = 673) or the "Veluwe" (n = 241). A binormal mixture model was fitted to the log-transformed optical density values for wild boar up to 20 months old to estimate the optimal cut-off value (-0.685) and accompanying sensitivity (90.6%) and specificity (93.6%). The overall seroprevalence was estimated at 24.4% (95% CI: 21.1-27.7%). The prevalence did not show variation between sampling years or regions, indicating a stable and homogeneous infection pressure from the environment. The relation between age and seroprevalence was studied in two stages. Firstly, seroprevalence by age group was determined by fitting the binary mixture model to 200 animals per age category. The prevalence showed a steep increase until approximately 10 months of age but stabilized at approximately 35% thereafter. Secondly, we fitted the age-dependent seroprevalence data to several SIR-type models, with seropositives as infected (I) and seronegatives as either susceptible (S) or resistant (R). A model with a recovery rate (SIS) was superior to a model without a recovery rate (SI). This finding is not consistent with the traditional view of lifelong persistence of T. gondii infections. The high seroprevalence suggests that eating undercooked wild boar meat may pose a risk of infection with T. gondii.

The Q fever outbreak in the Netherlands in 2007-2010 prompted government interventions to reduce the human incidence by reduction of Q fever shedding at dairy goat farms. Mandatory hygiene measures were taken, including the control of animal reservoirs. It has been postulated that brown rats, through their commensal nature, form an important factor in the persistent dissemination of endemic circulating Coxiella burnetii in nature to domestic animals, livestock and humans. Here, the occurrence of C. burnetii in rats captured at different types of location during the Q fever outbreak in the Netherlands, viz. urban areas, nature areas and various types of farm has been determined. This is a first step towards the elucidation of the reservoir status of rats in veterinary and human Q fever epidemiology. C. burnetii DNA was detected in the spleen of 4.9% of the brown rats (Rattus norvegicus) and 3.0% of the black rats (Rattus rattus). Evidence for C. burnetii infection was also found in liver, kidney, lung and intestinal tissue but not in heart, brain and pancreas. C. burnetii IgGs were detected in 15.8% of the brown rats. Positive rats were collected at goat, pig, cattle and poultry farms, and urban locations; including locations outside the designated 5km "increased-risk" zones around bulk milk positive goat farms. The percentage of rat-positive locations was the highest for goat farms (50%) and cattle farms (14.3%). The presence of actively infected rats outside the lambing season and at multiple environmental settings including urban locations might suggest that rats are not merely a spill-over host due to infection by a contaminated environment but might represent true reservoirs, capable of independent maintenance of C. burnetii infection cycles and thereby contributing to spread and transmission of the pathogen. If frequent (re)introduction of C. burnetii to small ruminant farms can be caused by rats as maintenance reservoirs, mandatory wildlife control and lifelong vaccination of herds will be necessary.

Toxoplasma gondii is an important foodborne pathogen, and the cause of a high disease burden due to congenital toxoplasmosis in The Netherlands. The aim of this study was to quantify the relative contribution of sheep, beef and pork products to human T. gondii infections by Quantitative Microbial Risk Assessment (QMRA). Bradyzoite concentration and portion size data were used to estimate the bradyzoite number in infected unprocessed portions for human consumption. The reduction factors for salting, freezing and heating as estimated based on published experiments in mice, were subsequently used to estimate the bradyzoite number in processed portions. A dose–response relation for T. gondii infection in mice was used to estimate the human probability of infection due to consumption of these originally infected processed portions. By multiplying these probabilities with the prevalence of T. gondii per livestock species and the number of portions consumed per year, the number of infections per year was calculated for the susceptible Dutch population and the subpopulation of susceptible pregnant women. QMRA results predict high numbers of infections per year with beef as the most important source. Although many uncertainties were present in the data and the number of congenital infections predicted by the model was almost twenty times higher than the number estimated based on the incidence in newborns, the usefulness of the advice to thoroughly heat meat is confirmed by our results. Forty percent of all predicted infections is due to the consumption of unheated meat products, and sensitivity analysis indicates that heating temperature has the strongest influence on the predicted number of infections. The results also demonstrate that, even with a low prevalence of infection in cattle, consumption of beef remains an important source of infection. Developing this QMRA model has helped identify important gaps of knowledge and resulted in the following recommendations for future research: collect processing-effect data in line with consumer style processing and acquire product specific heating temperatures, investigate the presence and concentration of viable bradyzoites in cattle, determine the effect of mincing meat on bradyzoite concentrations using actual batch sizes, and obtain an estimate of the fraction of meat that has been frozen prior to purchase. With more accurate data this QMRA model will aid science-based decision-making on intervention strategies to reduce the disease burden from meatborne T. gondii infections in The Netherlands.

Following an outbreak of autochthonous canine babesiosis in the Netherlands, a request made to veterinarians and the public to collect ticks from companion animals resulted in 4298 ticks submitted between July 2005 and October 2006 to our center. Ticks were identified as Ixodes ricinus adults (2907/4298, 67.6%), Ixodes sp. nymphs (529/4298, 12.3%) and Ixodes sp. larvae (385/4298, 9.0%), I. hexagonus adults (328/4298, 7.6%), Dermacentor reticulatus (72/4298, 1.7%), and several other exotic tick species such as Amblyomma flavomaculatum (formerly Aponomma flavomaculatum), Hyalomma marginatum rufipes, Rhipicephalus sanguineus, and R. turanicus (55/4298, 1.3%). Eight localities were surveyed for the presence of local D. reticulatus, a tick not indigenous to the Netherlands, based on multiple submissions of D. reticulatus ticks from these areas. D. reticulatus was collected from the vegetation in six of these localities, confirming the presence of populations of this tick in the Netherlands. Adult I. ricinus (n=251), I. hexagonus (n=237), and D. reticulatus (n=344) ticks were selected at random and subsequently screened by polymerase chain reaction (PCR) and reverse line blot (RLB) hybridization for the presence of Borrelia, Babesia, Theileria, Anaplasma, Ehrlichia, and Rickettsia species. I. ricinus ticks were infected with Rickettsia helvetica (24.7%), spirochetes belonging to the Borrelia burgdorferi sensu lato group (7.2%), the Ehrlichia-like "Schotii" variant (2.4%), Anaplasma phagocytophilum (1.6%), Babesia sp. (EU1) (1.2%), Babesia divergens (0.4%), and Babesia microti (0.4%). A. phagocytophilum (5.9%) and R. helvetica (0.8%) were also detected in adult I. hexagonus ticks. Spotted fever group Rickettsiae, previously reported as Rickettsia sp. DnS14/RpA4 (14.0%), and Borrelia burgdorferi sensu lato (0.3%) were detected in the D. reticulatus ticks, which appeared to be free from B. canis infection. We concluded that a much broader spectrum of ticks and tick-borne pathogens is present in the Netherlands than previously thought, including several potential zoonotic pathogens.