Canine filariasis

Veterinary Parasitology 77 (1998) 267±275

Canine filariasis Importance and transmission in the Baix Llobregat area, Barcelona (Spain) Carlos Arandaa,*, Octavi Panyellab, Roger Eritjaa, Joaquim CastellaÁb a

Servei de Control de Mosquits, Consell Comarcal del Baix Llobregat, Parc Torreblanca, CN 340 s/n, Sant Feliu del Llobregat 08980, Spain b Departament de Parasitologia, Facultat de VeterinaÁria, UAB, 08193 Bellaterra, Spain Received 10 July 1997; accepted 14 January 1998

Abstract Field and laboratory studies were performed in order to assess the degree of canine dirofilariasis caused by Dirofilaria immitis (Leidy) in the Baix Llobregat region, a fluvial area near Barcelona, Spain. A total of 188 dogs were sampled between May and August of 1994. Three main areas were chosen: the Western Delta, the Eastern Delta and the Northern zone. Simultaneously, a mosquito sampling programme was carried out with CO2 light traps, to search for infective larvae (L3) of D. immitis. Of the 188 dogs sampled, 38 were positive for at least one of the three filaria found: D. immitis 12.8%, Dipetalonema reconditum (Grassi) 3.7% and Dipetalonema dracunculoides (Cobbold) 2.7%. Only 1.1% showed a mixed infection of both D. immitis and D. dracunculoides. Although Dirofilaria repens Raillet et Henry has been found in Spain, it was not found in this study. Comparing the three zones of the Baix Llobregat, the Eastern Delta showed the highest level of D. immitis (35.3%), probably due to the presence of Aedes caspius (Pallas). Despite the effort in sampling the mosquito population, D. immitis was not found in any of the 2001 females dissected, belonging to 5 species. # 1998 Elsevier Science B.V. Keywords: Diro®laria immitis; Culex pipiens; Aedes caspius; Dog; Spain

1. Introduction Dirofilaria immitis (Leidy), is a filariid worm which typically lives as adults in the right ventricle of the heart and the pulmonary arteries of the dog, causing the canine * Corresponding author. Fax: +34 3 630 01 42; e-mail: [email protected] 0304-4017/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 0 9 - 5

Fig. 1. Map of Spain and map of the Baix Llobregat with the three zones described: Western Delta, Eastern Delta and Northern Zone.

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heartworm disease. The parasite is widely dispersed and is found in the tropics, subtropics and temperate zones (Boreham and Atwell, 1985). Although adult D. immitis affects dogs, cats, foxes and other wild mammal (Jones et al., 1993; Haddock, 1987), larvae have been reported sporadically in humans from various parts of the world (Mimori et al., 1986; Yoshimura and Wescott, 1989) and, as Dirofilaria repens Raillet et Henry, it has been reported affecting humans in Spain (Fuentes et al., 1994; GoÂmez Bautista and Rojo VaÂzquez, 1990). In the last few decades, this filarial disease appears to be an emerging zoonosis of public health importance in different parts of the world (Genchi et al., 1992; Russell and Geary, 1992). The distribution and prevalence of D. immitis and other vector-borne filariae in Spain are not well known, related in most cases to clinical studies but not to field work (Guerrero et al., 1989) and even underestimated (GutieÂrrez Galindo et al., 1995). Prevalences found in this country range from 0% to 37% with wide areas free of disease (Rojo VaÂzquez and GoÂmez Bautista, 1993). Several species of mosquitoes have been involved in the transmission of filaria (Macdonald, 1971), but only a few have been proven to transmit infective third stage of D. immitis (Hendrix et al., 1986). In Europe, Culex pipiens Linnaeus, Aedes caspius (Pallas) and Culex theileri Theobald have been implicated among other species that include the genus Anopheles (Ribeiro et al., 1983; Genchi et al., 1992). Although some species have been incriminated in the transmission of filaria in Spain (PeÂrez-SaÂnchez et al., 1989; Cordero del Campillo et al., 1994), no work has been carried out in this country to assess the vectorial status of the major mosquito species. In this project, field and laboratory studies were performed in order to show the prevalence of canine filariasis in the Baix Llobregat region, an agricultural and fluvial area near Barcelona, Spain (Fig. 1). The study assesses the number and distribution of rural dogs infected and the species composition of the mosquitoes in the region. 2. Material and methods 2.1. Blood samples A total of 188 local, rural dogs were sampled between May and August of 1994 from the three areas into which the Baix Llobregat was divided: the Western Delta, the Eastern Delta and the Northern zone (Fig. 1). All dogs found in the field were sampled. The dogs were living near to different mosquito habitats of the area. They were immobilized with the cooperation of the owner for the withdrawal of blood samples. At least 2 ml of blood were drawn from the cephalic vein with sodium citrate vacuum tubes. All samples were obtained during the day. Modified Knott tests were performed within three days of the survey. Blood was hemolysed with 10 ml of 2% formalin and centrifuged at 2500 rpm for 5 min, the supernatant was decanted, and the sediment was stained with a drop of methylene blue 0.1%. For differentiation of microfilariae (mff), positive Knott samples were further analyzed using the distribution of acid phosphatase (Chalifaux and Hunt (1971). Samples were also analyzed with a commercial ELISA assay, the Snap Canine Heartworm Antigen Test (Idexx Corporation,

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Maine, USA) for antibodies against adult antigens of D. immitis and to confirm Knott results. 2.2. Mosquito catches Between April and October, a weekly mosquito sampling programme was carried out with all night CO2 baited light traps (EVS trap, Bioquip, Gardena, CA, USA), to search for infective larvae (L3). Traps were located in 16 stations throughout the entire region and resulted in 326 catches. The mosquito habitats consisted mainly of polluted ditches, salt marshes in the eastern delta, and few spring creeks in the northern zone. As it can be deduced from the number of catches, it was impossible to obtain all weekly samples at some stations due to malfunction of the traps or to vandalism. The mosquitoes were taken to the laboratory, anaesthetized by chilling, identified and examined for L3. Two different procedures were used, taking into account the number of mosquitoes caught. In small samples, the head, thorax and abdomen of each female were separated on a glass slide, teased apart in a drop of PBS and then examined at 100. Large samples were examined using the technique described by Scoles et al. (1993). Each mosquito was placed in a well of a 24-well tissue culture plate and carefully decapitated with insect pins. Each well contained 1 ml of PBS. The plates were stored in a 378C incubator for 90 min to allow L3 to migrate into the medium. Plates were examined at 40 magnification on an inverted microscope. 3. Results Microfilariae were detected in 38 (20.2%) of the 188 dogs sampled, and classified as belonging to three different species: Dirofilaria immitis, Dipetalonema reconditum (Grassi) and Dipetalonema dracunculoides (Cobbold). Almost all dogs were used as watchdogs living in open air and were in poor condition. Both sexes were well represented in the region (43% m, 57% f). The exact age of the dogs was impossible to evaluate because of the lack of information and interest from most of the owners. However, it was possible to evaluate if the animal was more than 5 years old or not. Results are given in Table 1. In the study area, 53.3% of the dogs were older than 5 and 44.7% younger. Analysis of the data by the chi-square goodness of fit test, shows Table 1 Distribution of surveyed dogs by age and sex for each area sampled Western Delta

Eastern Delta

Northern zone

Total

m

f

m

f

m

f

m

f

<5 years >5 years Total by sex

33 22 55

18 15 33

11 15 26

13 12 25

16 11 27

11 11 22

60 48 108

42 38 80

Total

88

Sex

51

49

188

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Table 2 Number and percentage in brackets of dogs infected by different filariids in the three areas D. immitis Western Delta 3 (3.4) Eastern Delta 18 (35.3) Northern Zone 1 (2) Total 22 (11.7)

D. D. immitis ‡ D. dracunculoides dracunculoides

D. reconditum

Doubtful

Negative

3 (3.4) 0 0 3 (1.6)

1 5 1 7

2 (2.3) 0 2 (4.1) 4 (2.1)

77 28 45 150

2 (2.3) 0 0 2 (1.1)

(1.1) (9.8) (2) (3.7)

Sampled dogs

(87.5) 88 (54.9) 51 (91.8) 49 (79.8) 188

that differences in sex and age were only statistically important (P<0.05) in the Western Delta where more males and young individuals were sampled. Results of infection by zones and species are given in Table 2. In the whole area, D. immitis mff were found in 24 (12.8%) cases, D. reconditum in 7 (3.7%), and D. dracunculoides in 5 (2.7%). Two mixed infections of D. immitis and D. dracunculoides were detected. Four dogs contained parasites which could not be identified to species. All confirmed cases were positive for Knott and acid phosphatase activity identification and positive for Snap Canine Heartworm Antigen in D. immitis infections, except three that were identified only by positive Snap. Prevalence varied by zone (P<0.05) with the Eastern Delta as the zone with highest prevalence of mff in dogs, especially by D. immitis (35.3%). Differences in prevalence by sex for the total of cases were not statistically significant (P>0.1) although difference in age was an important factor (P<0.05) with 73% of old dogs positive for mff (data not shown). All 4 doubtful cases were negative on Snap and positive on the Knott test. For technical reasons, microfilariae were impossible to identify in phosphatase activity. We decided to classify them as doubtful, due to the impossibility of confirming the species. Members of the genera Culex, Aedes and Culiseta were collected during the study. CDC trap collections caught 2245 mosquitoes, namely 2120 Culex pipiens, 71 Aedes caspius, 32 Ae. vexans (Meigen), 14 Culiseta longiareolata (Macquart) and 8 Culiseta subochrea (Edwards) (Table 3). Culex pipiens was the most abundant mosquito in all Table 3 Number of mosquitoes captured per month and number of dissected mosquitoes. In brackets percentages for each species referred to the total mosquitoes captured

May June July August September October Total Total dissected

Cx. pipiens

Ae. caspius

Ae. vexans

Cs. longeareolata

Cs. subochrea

Total

129 673 405 275 467 171 2,120 (94.4) 1,901

0 0 0 0 0 71 71 (3.2) 52

0 23 4 0 0 5 32 (1.4) 32

6 4 3 0 1 0 14 (0.6) 14

0 3 0 3 2 0 8 (0.4) 2

135 703 412 278 470 247 2245 2001

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traps and in the study throughout the season while Ae. caspius and Ae. vexans appeared only in some occasions after local floods in June and October. A total of 2001 mosquitoes, 89% of the total trapped, were examined for infective L3. No L3 filaria was found in any of the individuals. 4. Discussion and conclusions There was a high prevalence of D. immitis in the dog population sampled. The total percentage of infected dogs, 12.8%, can be compared with data obtained in other parts of Spain. In Salamanca, an irrigated agricultural area similar to the Baix Llobregat, PeÂrezSaÂnchez et al. (1989), found 12.3% of dogs infected. Anguera (1995), found in rice plains in the Ebro Delta, more than double, 33.7%. A similar method was followed in all these studies. Free-ranging dogs were sampled that were usually found living in the open air. In contrast, surveys made asking veterinarians to cooperate by sampling dogs from their own consulting room gave much lower results, ranging in the Barcelona district from 0.6% (Guerrero et al., 1989), 1.2% (Rojo-VaÂzquez et al., 1990) to 3.6% (GutieÂrrez Galindo et al., 1995). This fact could be explained by the behavior of dog owners that live in the country, who do not frequent veterinarians. As might be expected, these dogs, more exposed to mosquito bites, have an increased probability of being infected by D. immitis. Haddock (1987) in his review of the distribution of canine heartworm disease, reported figures of more than 50% and even 70% in Japan, USA and Canada, markedly higher than the 36.7% found in Huelva, which is the highest percentage obtained in Spain (Guerrero et al., 1989). In the Mediterranean Basin, in Italy, Tassinari and Bonsembiante (1984) reported percentages ranging from 3.8% to 30.2%. The prevalence in Portugal reaches in some areas 91.2% (Ribeiro et al., 1983). Although D. repens has been found in Spain (PeÂrez-SaÂnchez et al., 1989; RojoVaÂzquez et al., 1990; Anguera, 1995), it was not found in this study. The absence of dogs infected with D. repens was unexpected. This mosquito and flea-borne filariid (PeÂrezSaÂnchez et al., 1989) has been reported, in similar areas, with a moderate prevalence (9.4% in the Ebro Delta). Dipetalonema reconditum is transmitted by fleas and lice (Georgi and Georgi, 1992). It has been found in similar numbers in Salamanca and Barcelona although it was not found in the Ebro Delta. Dipetalonema dracunculoides, a tick-borne filariid, has been found in Barcelona and Ebro Delta, but not in Salamanca (PeÂrez-SaÂnchez et al., 1989; Rojo-VaÂzquez et al., 1990; Anguera, 1995). This spotted distribution may be due to potential vector distribution. As shown in Table 2, the highest percentage of D. immitis mf was found in the Eastern Delta (35.3%).The coastal marshes of this area allow Ae. caspius to breed during local floods that generally occur in spring and fall. This species is rarely found in other zones of the Baix Llobregat. In the Northern zone, where studies carried out by the Baix Llobregat Mosquito Control Service (MCS) show very low mosquito densities, also has low densities of dogs. Only one case of D. immitis was found. Of the total of 24 infected dogs, 14 were males, confirming the absence of significant sex ratio differences obtained in Spain and other countries (Haddock, 1987;

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Rojo VaÂzquez and GoÂmez Bautista, 1993). In contrast, the association of age with the presence of filariid infection is markedly significant, with 20 of the 24 dogs infected with D. immitis (and 30 of the 38 total reported infected cases) being older than 5. Culex pipiens was the most abundant species collected followed by Ae. caspius and Ae. vexans in concordance with data from the MCS. Culiseta longiareolata and Cs. subochrea are species that occur in low numbers in adult captures made by this agency. Culex pipiens, the main mosquito in the study with 94% of the captures, has been incriminated as a good vector for certain reasons. The nocturnal feeding activity of this species, when circulating mff are at the lowest numbers in the peripheral blood of the host, can insure a greater chance of survival for the female due to the intake of minimal number of mff, which in large numbers can cause a high mortality (Haddock, 1987; Apperson et al., 1989). Coluzzi and Trabucchi (1968), found that mff passing through the foregut of Culex mosquitoes after ingestion are subjected to mechanical damage by the teeth of cibarial and pharyngeal armature. The authors stated thus that Cx. pipiens would appear to be a more efficient vector as this mosquito suffers only slight damage caused by a few numbers of filaria and can transmit the infection to a new host efficiently. Of the 2001 mosquitoes dissected not a single one had any L3 filaria. The reason may be the relatively low number of mosquitoes dissected, which is in concordance with some other studies. Ribeiro et al. (1983), found only L1 and L2 in one Cx. theileri of the total 554 mosquitoes dissected, and not one L3. Afolabi et al. (1989), did not find any L3 in 144 Culex erraticus (Dyar and Knab) captured by CO2 traps. Lowrie (1991), reported some studies that range from no L3's recovered in 10,000 Cx. quinquefasciatus dissected to 13 L3's in 2312 mosquitoes of the same species. Only six L3's were recovered in a study carried out on 2885 mosquitoes in North Carolina (Parker, 1986). Aedes caspius can be a potential vector of heartworm infection although it has been considered a non-resistant host as blood with high microfilaremias can affect the activity of the mosquito due to the incapability of avoiding the intake of large numbers of mff, which can cause a high mortality (Genchi et al., 1992). Despite these data and those from density populations that indicate Cx. pipiens as a better potential vector than Ae. caspius, higher numbers of infected dogs were found in the Eastern Delta where this species breeds together with Cx. pipiens. Although only 71 Ae. caspius were caught during the study, this number can be dramatically increased depending on the year. The extension of the Ae. caspius floodwater habitats depends on the number and severity of floods that occur, producing a very high mosquito population. The year of the study was especially dry, potentially reducing Ae. caspius populations density. Because dogs over 5 years of age were most commonly infected by D. immitis, they could have been infected during their lifetime by the bites of mosquitoes from prior years. Although Ae. vexans, the third most numerous mosquito, has been considered an important vector of canine filariasis, only one infection was found in the Northern zone, where this species was trapped.

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