Elena Pinelli

Zoonotic parasites in fecal samples and fur from dogs and cats in The Netherlands

Pets may carry zoonotic pathogens for which owners are at risk. The aim of the study is to investigate whether healthy pets harbour zoonotic parasitic infections and to make an inventory of the interactions between pet-owners and their companion animals in The Netherlands. Fecal and hair samples were collected from healthy household dogs and cats in Dutch veterinary practices. Owners were interviewed about interaction with their pets. The samples were investigated by microscopy, ELISA, and PCR. From 159 households, 152 dogs (D) and 60 cats (C), information and samples were collected and examination for several zoonotic parasites was performed. Toxocara eggs were found in 4.4% (D) and 4.6% (C) of the fecal samples and in 12.2% (D) and 3.4% (C) of the fur samples. The median epg in the fur was 17 (D) and 28 (C) and none of these eggs were viable. From 15.2% of the dog and 13.6% of the cat feces Giardia was isolated. One canine and one feline Giardia isolate was a zoonotic assemblage A (12%). Cryptosporidium sp. were present in 8.7% (D) and 4.6% (C) of the feces. Fifty percent of the owners allow the pet to lick their faces. Sixty percent of the pets visit the bedroom; 45-60% (D-C) are allowed on the bed, and 18-30% (D-C) sleep with the owner in bed. Six percent of the pets always sleep in the bedroom. Of the cats, 45% are allowed to jump onto the kitchen sink. Nearly 39% of the dog owners never clean up the feces of their dog. Fifteen percent of the dog owners and 8% of the cat owners always wash their hands after contact with the animals. Close physical contact between owners and their pets is common and poses an increased risk of transmission of zoonotic pathogens. Education of owners by the vet, specifically about hygiene and potential risks, is required.

High Malnutrition Rate in Venezuelan Yanomami Compared to Warao Amerindians and Creoles: Significant Associations WITH Intestinal Parasites and Anemia

Background Children in rural areas experience the interrelated problems of poor growth, anemia and parasitic infections. We investigated the prevalence of and associations between intestinal helminth and protozoan infections, malnutrition and anemia in school-age Venezuelan children. Methods This cross-sectional study was conducted in 390 children aged 4-16 years from three rural areas of Venezuela: the Amazon Region, Orinoco Delta and Carabobo State. Stool samples were collected for direct parasitic examinations. Anthropometric indicators of chronic (height-for-age Z score) and acute (weight-for-height and Body Mass Index (BMI)-for-age Z score in respectively children under 5 years of age and children aged 5 years and above) malnutrition were calculated. Multivariate linear and logistic regression models were built to determine factors associated with nutritional status and polyparasitism. Results Hookworm and Strongyloides stercoralis prevalences were highest in children from the Amazon rainforest (respectively 72% and 18%) while children from the Orinoco Delta and Carabobo State showed higher rates of Ascaris lumbricoides (respectively 28% and 37%) and Trichuris trichiura (40% in both regions). The prevalence of Giardia lamblia infection was not significantly different between regions (average: 18%). Anemia prevalence was highest in the Amazon Region (24%). Hemoglobin levels were significantly decreased in children with a hookworm infection. Malnutrition was present in respectively 84%, 30% and 13% of children from the Amazon Region, Orinoco Delta and Carabobo State. In multivariate analysis including all regions, G. lamblia and helminth infections were significantly and negatively associated with respectively height-for-age and weight-for-height/BMI-for-age Z scores. Furthermore, hemoglobin levels were positively associated with the height-for-age Z score (0.11, 95% CI 0.02 - 0.20). Conclusions In rural populations in Venezuela helminthiasis and giardiasis were associated with acute and chronic nutritional status respectively. These data highlight the need for an integrated approach to control transmission of parasites and improve the health status of rural Venezuelan children.

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity.

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.

Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

Evaluation and improvement of two PCR targets in molecular typing of clinical samples of Leishmania patients

Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.

Molecular cloning and sequencing of the cDNA for dog interleukin-4

T lymphocytes play a crucial role in the development of protective immunity against intracellular pathogens. Studies on mouse CD4 T cell clones showed that helper T (Th) cells can be divided into Th1 and Th2 subsets based on their cytokine production. Interleukin-4 (IL-4) is a cytokine produced predominantly by the Th2 subset. It is involved in stimulation and class switching of B cells, acts as a growth factor for Th2 cells, and inhibits Th1 type of reactivity (Mosmann et al. 1987). In mice resistance and susceptibility toLeishmania infection were highly correlated with predominance of Th1 and Th2 reactivity, respectively (Locksley and Scott 1991). We study the immune responses in dogs infected with Leishmania infantum. Protective immunity in canine visceral leishmaniosis (CVL) is associated with a Th1 type of response (Pinelli et al. 1994, 1995). Our knowledge of CVL would greatly benefit from the development of dog Th2 specific reagents. This paper reports the cloning and sequencing of cDNA containing the coding sequence of dog IL-4 performed by the rapid amplification of cDNA ends (RACE) protocol (Frohman 1995). For this purpose mRNA isolated from concanavalin-A-stimulated dog peripheral blood mononuclear leucocytes isolated from a Leishmania–infected dog was reverse transcribed into

Complement evasion by Bordetella pertussis: implications for improving current vaccines

Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.

Helminths and skewed cytokine profiles increase tuberculin skin test positivity in Warao Amerindians

The immune regulatory mechanisms involved in the acquisition of Mycobacterium tuberculosis infection in children are largely unknown. We investigated the influence of parasitic infections, malnutrition and plasma cytokine profiles on tuberculin skin test (TST) positivity in Warao Amerindians in Venezuela. Pediatric household contacts of sputum smear-positive tuberculosis (TB) cases were enrolled for TST, chest radiograph, plasma cytokine analyses, QuantiFERON-TB Gold In-Tube (QFT-GIT) testing and stool examinations. Factors associated with TST positivity were studied using generalized estimation equations logistic regression models. Of the 141 asymptomatic contacts, 39% was TST-positive. After adjusting for age, gender and nutritional status, TST positivity was associated with Trichuris trichiura infections (OR 3.5, 95% CI 1.1-11.6) and low circulating levels of T helper 1 (Th1) cytokines (OR 0.51, 95% CI 0.33-0.79). Ascaris lumbricoides infections in interaction with Th2- and interleukin (IL)-10-dominated cytokine profiles were positively associated with TST positivity (OR 3.1, 95% CI 1.1-8.9 and OR 2.4, 95% CI 1.04-5.7, respectively). A negative correlation of QFT-GIT mitogen responses with Th1 and Th2 levels and a positive correlation with age were observed (all p < 0.01). We conclude that helminth infections and low Th1 cytokine plasma levels are significantly associated with TST positivity in indigenous Venezuelan pediatric TB contacts.

Increase of imported Leishmaniasis in the Netherlands: a twelve year overview (1996–2007)

Surveillance data indicates that the number of cutaneous (CL), mucocutaneous (ML) and visceral leishmaniasis (VL) cases has increased globally and in Europe during the past decades. Leishmaniasis is only seen as an imported disease in the Netherlands. We investigated occurrence in the Netherlands through an analysis of Leishmania patients sent to our laboratory and to the nationwide network of the pathology departments between 1996 and 2007. The majority of patients suffered from CL, and an outbreak among military personnel stationed in endemic regions in 2005 was noted. ML was rarely found. However, the occurrence of VL has clearly increased. Physicians in non-endemic regions should be aware that leishmaniasis can be contracted as close as Southern Europe and that it is not limited to tropical and subtropical regions only.

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.