Epidemiological survey of anti-flea IgE in dogs in Japan by using an antigen-specific IgE quantitative measurement method

In Japan, an epidemiological survey was performed in dogs from October to December 2008 by using a quantitative measurement method for antigen-specific IgE towards specific Ctenocephalides felis antigens. 214 dogs from 22 veterinary clinics were included. These clinics were located as follows, from North to South: Hokkaido, Aomori, Fukushima, Tochigi, Saitama, Chiba, Tokyo (Tama-City and Ota-ku), Kanagawa, Gifu, Niigata, Kyoto, Nara, Osaka, Hyogo, Kagawa, Ehime, Hiroshima, Yamaguchi, Fukuoka, Kumamoto and Kagoshima. 110 dogs (51.4%) were seropositive for flea-specific IgE. No differences were associated with gender or breed. This survey confirms that flea infestation in dogs is a common problem in Japan. It especially shows that the infestation also occurs in Northern Japan where fleas are considered uncommon by the vet.

F leas are the most common ectoparasites of both dogs and cats throughout the world (Beugnet & Franc, 2010;Farkas et al., 2009). Due to their ability to reproduce in households, fleas may be present all year round, even during winter season, which explains why dogs living in cold areas may be infested. Flea bites may induce pruritus and the major pathogenic effect is flea allergic dermatitis (FAD). FAD prevention is based on the control of the flea infestation, involving regular and continued use of anti-flea drugs, which are usually topical formulations but can also be given orally (Beugnet & Franc, 2010;Farkas et al., 2009;Masuda et al., 2002;Medleau et al., 2003). No reports on epidemiological surveys regarding flea infestations in dogs in Japan have been published. It is generally thought that the flea infestation is scarce in the Northern part of Japan due to its cold climate. Instead of performing flea counts in order to evaluate their prevalence in dogs, which is not always easy and takes time, the authors suggested a new method to assess the flea infestation by testing the specific serological response to flea allergens. Seropositivity is a valid proof of a flea bite, even if the time (recent or old infestation) as well as the degree of infestation cannot be established. Cat flea allergens are high-molecular proteins found in flea saliva (McDermott et al., 2000). Cat flea salivary extracts also show great antigenic diversity recognized by IgE from artificially sensitized dogs or from those with FAD (Lee et al., 1999). McDermott et al. cloned a major 18-kD allergen, called Cte f 1 in the international nomenclature (Medleau et al., 2003). Its structure is very similar to that of the original molecule in terms of flea IgE-binding (Medleau et al., 2003). Intradermal testing is the standard method for identifying IgE-mediated allergic reaction in vivo, but it requires specific equipment and manipulation in each veterinary clinic (Laffort-Dassot et al., 2004). At the opposite, specific serum IgE measurement can be performed in a well-equipped laboratory and requires only a blood sample at the veterinary clinic level. As for intradermo-reaction/prick tests, it is intended to reveal the current sensitization level to flea allergens. In this survey, serum concentration of antigenspecific IgE was quantitatively measured in ng/mL using a published technique (Okayama et al., 2010). The specificity of this technique has been previously Research note Parasite, 2012, 19, 173-176 controlled by using several antigens (flea and pollens), experimental dogs that were not sensitized and experimental dogs that were sensitized. The assay enables to monitor serum antigen-specific IgE concentrations in dogs, providing information on flea exposure (Fujimura et al., 2011).

MATERIALS AND METHODS
S erum concentration of IgE against flea antigens was measured in this epidemiological study in dogs in Japan. 22 veterinary clinics were recruited throughout Japan districts to get an estimation of flea burden on dogs (Fig. 1). All veterinary clinics were asked to randomly recruit ten dogs and collect a blood sample during the period from August to October 2008. The selected dogs had to be healthy and were not to show any clinical signs, especially dermatosis and pruritus. All dogs were client-owned and presented to the animal hospitals for routine check-ups. The age of the dogs ranged from two to seven years. A total of 214 blood samples were collected. The profiles of the dogs examined are shown in Table I. Serum samples from dogs were collected from October to December 2008 in the following 22 areas: Hokkaido, Aomori, Fukushima, Tochigi, Saitama, Chiba, Tokyo (Tama-City and Ota-ku), Kanagawa, Gifu, Niigata, Kyoto, Nara, Osaka, Hyogo, Kagawa, Ehime, Hiroshima, Yamaguchi, Fukuoka, Kumamoto and Kagoshima (Fig. 1)  ries, Inc (Kanagawa, Japan), according to a procedure previously published (Okayama et al., 2010). In short, 40 kinds of allergen-IgE including 22 environmental allergens and 18 food allergens were measured in this assay. The antigen of flea whole body was purchased from Greer Laboratories, Inc. (Lenoir, NC, USA) and used in this assay. Prior to the study, flea-specific IgE concentrations were measured in four experimental healthy Beagle dogs (two females of five-month-old and two males of four-month-old) kept in a flea-freecontrolled facility. The values obtained were respectively 0, 3, 2 and 13 ng/mL, with an average of 4.5 and a standard deviation of 5.8. The cut off was calculated as mean + 2 SD = 16 ng/mL. Then, based on previously published (McDermott et al., 2000) assessments, we estimated flea IgE positive when data showed > 16 ng/mL for flea IgE. The total IgE half-life is considered to be short (12 hours in mice to two days in humans) (Hirano et al., 1983;Tizard, 2009). However, IgE production from plasma cells seemed to continue for several months and serum antigen-specific IgE was detected during off-season of offending allergen (Masuda et al., 2002;Tizard, 2009). It was therefore hypothesized that a positive result would indicate a current or past flea exposure with no possibility to a precise estimation of the flea infestation status.
Cross-reactivity of IgE against house dust mites may influence flea-IgE titers. Therefore, all the cases with coincidental sensitization to house dust mites, i.e. when the IgE against house dust mites (Dermatophagoides farinae and/or Dermatophagoides pteronyssinus) was higher than flea-IgE, were excluded from this study. The statistical analysis used was the Steel-Dwass' test with IgE values according to gender, breed (if, n > 10) and area with a significance level of p < 0.05. This survey was undertaken from October to December 2008, which only covers the end of the entire flea season. As it was not conducted throughout the whole year, a season analysis per region could not be conducted.

RESULTS
T able I shows the results by gender and breed.   (Table II).

DISCUSSION
I n all, 51.4 % of dogs living in Japan were seropositive for anti-flea IgE meaning that they were either infested by fleas several weeks prior to survey dates or that they remained infested. As expected, we did not find any significant differences in gender and breed. Significant differences were present among areas, but the interpretation of the data is difficult due to the low number of samples. It will be necessary to include more dogs in a future study, to collect samples throughout a whole year in order to evaluate the seropositivity during the four seasons, and to take into account the impact of regular or occasional anti-flea treatments. In this time-limited study, we confirmed that dogs were often infested and bitten by fleas in Japan. We found solid proof that fleas are present in cold Northern areas, through the positivity rate of dogs that haven't travelled around the country, despite the fact that vets and pet owners believe that fleas cannot be found in those regions. Climate change may explain the higher flea prevalence in the North, as it has also been shown for other insects. In Hokkaido, cockroaches and beetles were rarely found several decades ago but they have now increased significantly in numbers (Kida, 2007). This survey also highlights the importance of controlling fleas, given that more than 50 % of dogs were infested with them. Future surveys will assess the impact of monthly flea treatments on this serological status.