Prevalence of Dirofilaria immitis (Nematoda: Filarioidea) in

VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION

Prevalence of Dirofilaria immitis (Nematoda: Filarioidea) in Mosquitoes From Northeast Arkansas, the United States TANJA MCKAY,1,2,3 T. BIANCO,1 L. RHODES,1

AND

S. BARNETT4

J. Med. Entomol. 50(4): 871Ð878 (2013); DOI: http://dx.doi.org/10.1603/ME12197

ABSTRACT A mosquito survey was conducted to identify which species of mosquitoes carry Dirofilaria immitis (Leidy) (Nematoda: Filarioidea), dog heartworm, in northeast Arkansas. Using polymerase chain reaction, mosquitoes were analyzed for D. immitis, Dirofilaria repens Railliet & Henry, and Acanthocheilonema dracunculoides Cobbold. Mosquitoes were collected from April to October 2009 using black light ultraviolet traps baited with dry ice. Sixteen mosquito species were identiÞed. D. immitis was identiÞed in nine mosquito species, which included Aedes vexans (Meigen), Anopheles quadrimaculatus Say, Anopheles punctipennis (Say), Culex pipiens quinquefasciatus Say, Culex erraticus (Dyer & Knab), Culiseta inornata (Williston), Psorophora columbiae (Dyer & Knab), Psorophora ferox (Humboldt), and Psorophora howardii Coquillett. No D. repens or A. dracunculoides DNA was ampliÞed. Of the 1,212 mosquito pools tested, 7.3% were positive for D. immitis. Frequency of D. immitis infections from six collection sites ranged from 2.1 to 19.4%. Ae. vexans and An. quadrimaculatus were the two most abundant species, composing 58.7 and 23.7% of the total mosquitoes collected, with 9.6 and 6.9% of pools positive for D. immitis, respectively. To investigate localized vector infection rates of D. immitis, mosquitoes were collected from inside the kennel of a heartworm-positive dog. Of the 114 mosquitoes collected, 84 (73.7%) were positive for D. immitis. The frequency of D. immitis-infected mosquitoes collected near a heartworm-positive dog was considerably higher than in the original six collection sites, suggesting a single heartworm-positive dog potentially increases infection pressure on susceptible animals sharing mosquito exposure. KEY WORDS Dirofilaria immitis, dog heartworm, Dirofilaria repens, mosquitoes, Arkansas

Dirofilaria immitis (Leidy) (Nematoda: Filarioidea), dog heartworm, is a Þlarial disease most associated with dogs, but can also be found in domestic cats and other felines (Bowman and Atkins 2009). This disease is vectored by mosquitoes (Ludlam et al. 1970) and has a cosmopolitan distribution (Comiskey and Wesson 1995). A considerable amount of attention has been given to heartworm disease in the United States, and multiple studies on mosquito vectors have been conducted in southern states such as Louisiana (Comiskey and Wesson 1995, Watts et al. 2001), Florida (Sauerman and Nayar 1983, Watts et al. 2001), and Georgia (Licitra et al. 2010). The highest prevalence of this disease is in the southeast, with high prevalence occurring along the Mississippi River (Bowman et al. 2009). Heartworm has been an important veterinary issue in the Mississippi Delta. In the 1940s and 1950s, Eyles et al. (1954) examined the prevalence of D. immitis infections in stray dogs in the City of Memphis and in 1 Department of Biological Sciences, Arkansas State University, P.O. Box 599, State University, AR 72467. 2 P.O. Box 2340, Arkansas Experiment Station, State University, AR 72467. 3 Corresponding author, e-mail: [email protected]. 4 Novartis Animal Health US, Inc., 3200 Northline Ave., Suite 300, Greensboro, NC 27408.

the surrounding Shelby Co., TN. They found 15.2% (618 dogs tested May 1944 through December 1945) and 20.6% (204 dogs tested September 1952 through January 1953) of the dogs infected with heartworm. Other canids are reported to harbor D. immitis. King and Bohning (1984) examined the hearts of coyotes, red foxes, and gray foxes from carcasses collected in northeast Arkansas (AR) and found coyotes to have heavier infections compared with foxes. Of the 193 coyotes examined, 127 (65.8%) were positive for D. immitis (King and Bohning 1984). The authors of that study concluded that coyotes likely serve as a significant reservoir for heartworm infections in northeast AR. To better understand the incidence in heartworm in pet dogs, Jones et al. (1993) surveyed AR veterinarians on the prevalence of dog heartworm. The areas along the Mississippi River (Delta Region) had a higher prevalence of D. immitis as compared with other areas in AR. Previously, the only known study examining mosquitoes for D. immitis in AR was performed in the city of Stuttgart by Jones and Meisch 1993. Although 1,060 Psorophora columbiae (Dyer & Knab) were dissected, no mosquitoes were infected with D. immitis. Since 2008, the Companion Animal Parasite Council (CAPC 2012) has been collecting data from commercial testing laboratories on canine heartworm and has

0022-2585/13/0871Ð0878$04.00/0 䉷 2013 Entomological Society of America

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JOURNAL OF MEDICAL ENTOMOLOGY

Vol. 50, no. 4

Fig. 1. Maps of the state of Arkansas and the city of Jonesboro with locations of the six mosquito-trapping locations (AÐF) and the collection site for the mosquitoes collected from the D. immitis-positive dog in Caldwell, AR (G). Jonesboro map redrawn from Map data 2012 Google.

prevalence maps available by county for each state in the United States (see prevalence maps: www. capcvet.org/parasite-prevalence-maps). In examining the 2008 data for AR, 19,576 dogs were tested for heartworm, of which 1,536 (7.8%) tested positive for D. immitis. The 2008 county data for AR (www. capcvet.org/parasite-prevalence-maps) show that Craighead County, located in northeast AR, had high prevalence, with 31.8% of all positive cases of heartworm in the state (of 3,729 dogs tested, 489 positive for D. immitis). D. immitis is now considered enzootic in canine populations and is spreading into new geographic locations (Bowman and Atkins 2009). Studies regarding mosquito vectors are important to understanding heartworm prevention. We therefore conducted the current study to identify potential mosquito vectors of heartworm in Jonesboro, a city located in Craighead County, AR. Mosquitoes were analyzed for the presence of DNA from D. immitis and two other nematodes, Dirofilaria repens Railliet & Henry and Acanthocheilonema dracunculoides Cobbold. We also examined the prevalence of D. immitis in mosquitoes that were collected inside a kennel of a dog that was known to be positively infected with dog heartworm and had never been on heartworm prophylaxis. Materials and Methods Mosquito Collections. Mosquitoes were collected from the Animal Control Shelter and Þve residential locations in the Jonesboro, AR, area (Fig. 1; Table 1). Mosquitoes were collected over 20 trap nights from 25 April to 19 October 2009 using New Standard Miniature blacklight ultraviolet (UV) traps (model 1212, John W. Hock Co., Gainesville, FL) baited with 1 kg dry ice. Traps were placed in the evening, around dusk, and operated for 24 hr. Mosquitoes were also

collected from around the property and from inside the kennel of a heartworm-positive dog located in Caldwell, AR, ⬇87.5 km south of Jonesboro (Fig. 1). The dog was a 5 yr-old female, black, American Pit Bull Terrier that had never been on heartworm prophylaxis. Collections, inside the kennel (4 by 4 m enclosure) near the dog, were made over 30 min on the following dates: 11 May, 8 Ð10 July, and 13Ð15 July 2009 using an AC insect vacuum (2820A, BioQuip Products Inc., Rancho Dominguez, CA) and aerial net. All mosquitoes were stored in the freezer at ⫺80⬚C until they were identiÞed using the key by Darsie and Ward (2005). Once identiÞed to species, the abdomens were separated from the head or thoraces to better understand their vector competency. Mosquito species positive in head or thoraces were presumed to harbor L3-stage larvae, the infective stage of D. immitis (Bradley and Nayer 1987). Mosquito pools positive for D. immitis only in the abdomen were considered L2 Table 1. collected Site no. A B C D E F G

A description of the locations where mosquitoes were

Longitude latitude 35⬚ 46⬘40.52⬙ N 90⬚ 42⬘42.47⬙ W 35⬚ 48⬘29.52⬙ N 90⬚ 45⬘06.29⬙ W 35⬚ 46⬘40.69⬙ N 90⬚ 43⬘20.23⬙ W 35⬚ 49⬘15.11⬙ N 90⬚ 37⬘05.74⬙ W 35⬚ 53⬘11.55⬙ N 90⬚ 41⬘05.87⬙ W 35⬚ 51⬘19.82⬙ N 90⬚ 47⬘28.04⬙ W 35⬚ 51⬘19.82⬙ N 90⬚ 47⬘28.04⬙ W

Description Urban, residential, borders Craighead Forest Park Urban, residential (Þve dogs in household) Urban, residential (dogs in neighborhood) Dog pound, rural (⬇5 dogs present at each visit, all outdoors) Rural, four dogs (outdoor) Rural/urban (25Ð30 dogs, all outdoor) Rural (Þve dogs on premise, one heartworm-positive dog)

July 2013

MCKAY ET AL.: PREVALENCE OF D. immitis IN MOSQUITOES

larvae, the noninfective stage of D. immitis (Ledesma and Harrington 2011). All mosquitoes were stored at ⫺80⬚C for further molecular analysis. DNA Extraction From Mosquitoes. Mosquito species were examined for D. immitis in pools of Þve heads or thoraces and abdomens. Mosquitoes collected from the heartworm-positive dog were analyzed individually. DNA extraction of mosquito specimens followed a modiÞed cetyl trimethylammonium bromideÐproteinase K method (SaghaiÐMaroof et al. 1984) with a phenolÐ chloroform extraction. The presence and quality of DNA was examined by agarose gel electrophoresis. Then samples were diluted to 10 ng/␮l after the quantity of DNA was determined using the NanoDrop ND-2000c Spectrophotometer (Thermo ScientiÞc, Waltham, MA). Polymerase Chain Reaction Amplification. An initial polymerase chain reaction (PCR) was applied using primers designed for the internal transcribed spacer region two (ITS2), more speciÞcally the 5.8SITS2Ð28S gene region. This primer pair has been referred to as DIDR-F1 and DIDR-R1 (Rishniw et al. 2006) or pan-filarial primers owing to the ability of the primer pair to amplify DNA for various nematodes at one time (Rishniw et al. 2006). The pan-filarial primers are speciÞcally designed to amplify Þlarial DNA from various nematodes, including D. immitis, Acanthocheilonema reconditum, D. repens, A. dracunculoides, and various species of Brugia (Rishniw et al. 2006). If mosquito pools were found to be positive for Þlarial DNA, additional PCRs were performed using three pairs of cytochrome oxidase subunit I (COI) primers. The Þrst COI primer pair used was D. immitis, named DI COI-F1 and DI COI-R1 (Rishniw et al. 2006). If D. immitis was not found, D. repens COI primers, named DR COI-F1 and DR COI-R1 (Rishniw et al. 2006), were used. A third primer pair, AR COI-F1 and AR COI-R1 (Rishniw et al. 2006), was used to detect A. reconditum, if samples were negative for D. immitis and D. repens. All PCRs were done in a BioRad iCycler thermal cycler (Hercules, CA) at 25 ␮l. Each PCR reaction contained a minimum of 10 ng/␮l of genomic DNA (1 ␮l), 12.5 ␮l GoTaqColorless Master Mix, 2X (Promega Corporation, Madison, WI), 1 ␮l of each of the primers (0.75 ␮M), and 9.5 ␮l nuclease-free water, in a Þnal reaction volume of 25 ␮l. A negative control was made by using sterile water instead of template DNA. A D. immitis-positive control was made using DNA from a laboratory strain of Aedes aegypti (L.) known to be infected with D. immitis (B.L. Blagburn, Auburn University). AmpliÞcations were carried out using an initial denaturation step at 94⬚C for 2 min, followed by 32 cycles, each consisting of denaturation for 30 s at 94⬚C, annealing for 30 s (at 60⬚C for pan-filarial primers, 58⬚C for the COI primers), and extension at 30 s at 72⬚C. The last cycle included a Þnal extension step at 72⬚C for 7 min and an end holding at 4⬚C. PCR product (5 ␮l) was examined on a 1.5% agarose gel electrophoresis and diagnostic PCR amplicons visualized and documented using a FOTO/Anaylst FX (Fotodyne Incorporated, Hartland, WI) UV transilluminator. The DIDR-F1 and

873

DIDR-R1 PCR primers yield a 542-, 578,- and 484-bp amplicon for D. immitis, A. reconditum, and D. repens, respectively (Rishniw et al. 2006). The sizes of the diagnostic amplicons for the COI primers were 203 (D. immitis), 209 (D. repens), and 208 bp (A. reconditum). After running the PCR products (pan-filarial and D. immitis) on a 1.5% agarose gel to conÞrm a positive reaction for both the pan-filarial and D. immitis primers, 25 ␮l of each PCR product for Anopheles quadrimaculatus Say, Aedes vexans (Meigen), Ps. columbiae, Culex pipiens quinquefasciatus Say, Culiseta inornata (Williston), and the positive control (Ae. aegypti) from B. Blagburn (Auburn University) were sent to the Arizona State University DNA Laboratory (Tempe, AZ) for sequencing. BLAST searches (http:// www.ncbi.nlm.nih.gov/BLAST) were applied to determine the species closest to the DNA sequencing results. All sequences ampliÞed with the pan-filarial primers were 91Ð100% identical to D. immitis (EMBLGenBank accession numbers: FJ263467; HM126607; EU087699). All sequences ampliÞed with speciÞc primers of D. immitis were 100 or 99% identical to D. immitis (EMBL-GenBank accession numbers: BQ454370; BQ455619; BQ457017). A ␹2 analysis was used to compare the proportions of pools positive for D. immitis in the head or thoraces versus in the abdomen for three species, Ae. vexans, Cx. p. quinquefasciatus, and An. quadrimaculatus. This statistical analysis was performed using Minitab release 16.1.0 (Minitab Inc. 2010, Minitab, Coventry, United Kingdom). Results In this study, 91,798 mosquitoes were collected, representing 16 species (Table 2). Ae. vexans and An. quadrimaculatus were the two most abundant species, composing of 58.7 and 23.7% of the total mosquitoes collected, respectively. Ae. vexans was the most abundant mosquito collected during April and May, with smaller numbers being observed in the summer months (Fig. 2). In July and August, there was a drastic shift in the distribution of species, with An. quadrimaculatus and the Culex spp. being the most abundant species. In total, 1,212 mosquito pools were examined using the pan-filarial primers (Table 3). D. immitis DNA was the only DNA ampliÞed, with 89 (7.3%) of the pools being positive. DNA from 13 species of mosquitoes was examined, of which nine species were found positive for D. immitis, which included Ae. vexans, An. quadrimaculatus, Anopheles punctipennis, Cx. p. quinquefasciatus, Culex erraticus (Dyer & Knab), Cs. inornata (Williston), Ps. columbiae, Psorophora ferox (Humboldt), and Psorophora howardii Coquillett. Of the nine species positive for D. immitis, four species had more than one pool positive, which included Cs. inornata, Cx. p. quinquefasciatus, Ae. vexans, and An. quadrimaculatus, with 25.0, 10.6, 9.6, and 6.9% of pools positive, respectively (Table 3). Of the Ae. vexans, Cx. p. quinquefasciatus, and An. quadrimaculatus mosquito pools that were positive for D. immitis,

874 Table 2. Location A B C D E F Ga Total % proportion


Vol. 50, no. 4

Total number of and species of female mosquitoes collected by site Mosquito species AA 26 18 5 14 1 0 0 64 0.07

AFP

AT

AV

AC

0 2 14,703 2 0 0 20,613 1 0 0 2,771 0 1 3 8,585 18 0 6 2,628 0 0 1 4,550 1 0 0 7 0 1 12 53,857 22 0.001 0.01 58.7 0.02

AP

AQ

27 7,871 10 6,155 12 2,718 33 2,539 8 87 3 1,870 0 508 93 21,748 0.1 23.7

CE

CPQ

1,007 153 1,439 204 529 73 3,100 2,210 80 54 1,232 271 17 1 7,404 2,966 8.1 3.2

CI

PC

PCi

PF

PH

US

31 199 0 7 4 51 16 104 15 22 4 3 12 153 0 2 1 4 12 2,130 17 283 14 15 15 20 0 1 0 1 2 2,054 0 0 0 0 0 35 0 0 0 0 88 4,695 32 315 23 74 0.1 5.1 0.03 0.3 0.03 0.08

T

U

0 61 0 20 0 39 3 89 0 55 0 137 0 0 3 401 0.003 0.4

Total 24,144 28,624 6,319 19,066 2,956 10,121 568 91,798 100

Notes: AA, Ae. albopictus; AFP, Aedes fulvus pallens; AT, Aedes triseriatus; AV, Ae. vexans; AC, Anopheles crucians; AP, An. punctipennis; AQ, An. quadrimaculatus; CE, Cx. erraticus; CPQ, Cx. pipiens quinquefasciatus; CI, Culiseta inornata; PC, Ps. columbiae; PCi, Ps. ciliata; PF, Psorophora ferox; PH, Ps. howardii; US, Uranotaenia sapphirina; T, Toxorhynchites; U, Unknown ⫽ unable to identify. a G, mosquitoes collected from around the property and inside the kennel of a heartworm-positive dog, Caldwell, AR.

the proportion of pools positive in the head or thorax versus in the abdomen varied among the three species (␹2 ⫽ 16.53, P ⬍ 0.000). For Ae. vexans, of the 53 positive pools, 38 pools were positive in the head or thorax (L3), whereas 15 were positive only in the abdomen (L2). For Cx. p. quinquefasciatus, eight mosquito pools were positive in the head or thorax (L3), whereas only one pool was positive in the abdomen (L2). In contrast, An. quadrimaculatus had more positive mosquito pools associated with the abdomen. Of the 20 positive pools for An. quadrimaculatus, 15 (75%) were associated with the abdomen (L2), whereas Þve were positive in the head or thorax (L3). Too few positive pools of Cs. inornata and Ps. columbiae were observed for statistical analysis. However, all the Cs. inornata and Ps. columbiae pools that tested positive for D. immitis were all in the L3 infective stage. Frequency of D. immitis infections from the six collection sites ranged from 2.1 to 22.4% (Table 3). Interestingly, the Animal Control Shelter (location D), where one would expect a high prevalence, as seen in the study by Tzipory et al. (2010), had the lowest proportions of positive pools. The highest prevalence of heartworm was found at location E, in the

northern part of Jonesboro, a more rural location with four outdoor dogs present. More positive samples were detected from mosquitoes collected in April (22.5%) as compared with samples collected from May to October (Table 4). Four species of mosquitoes were collected from inside the dog kennel housing a heartworm-positive dog, which included Ae. vexans, An. quadrimaculatus, Cx. erraticus, and Ps. columbiae. Of the 114 mosquitoes collected during the seven collection dates, 84 (73.7%) were positive for D. immitis (Table 5). Of the four mosquito species collected, An. quadrimaculatus and Cx. erraticus were the only two species positive for D. immitis. An. quadrimaculatus was the most abundant mosquito collected at the dog kennel, with 83.5% of the individuals positive for D. immitis. Although only seven individuals of Cx. erraticus were analyzed for D. immitis, three individuals were positive (42.9%). Of the seven collection dates where more than one mosquito was collected and examined for D. immitis, the percentage of D. immitis-positive mosquitoes collected ranged from 53.8 to 92.3%. Of the 81 An. quadrimaculatus positive for D. immitis, 44 (54.3%) individuals were found to have infections in their head or

Fig. 2. Total number of mosquitoes collected from 25 April to 17 October, 2009.

July 2013 Table 3.

MCKAY ET AL.: PREVALENCE OF D. immitis IN MOSQUITOES Number of mosquito pools tested and number of PCR pools positive for D. immitis in the Jonesboro, AR, area

Species Ae. albopictus Ae. triseriatus Ae. vexans Anopheles crucians An. punctipennis An. quadrimaculatus Culex erraticus Cx. p. quinquefasciatus Culiseta inornata Psorophora columbiae Ps. ferox Ps. howardii Uranotaenia sapphirina Total % positive a

875

Location A

B

C

D

E

F

2 (0) Ð 113 (9) Ð 4 (0) 62 (5) 14 (1) 15 (5) 1 (0) 4 (0) 2 (0) Ð 2 (0) 219 (20) 9.1

2 (0) Ð 114 (4) Ð 2 (0) 49 (3) 27 (0) 10 (1) 3 (2) 10 (0) 7 (1) Ð Ð 224 (11) 4.9

Ð Ð 64 (7) Ð 3 (0) 60 (8) 8 (0) 3 (0) 2 (0) 9 (1) 1 (0) Ð Ð 150 (16) 10.7

1 (0) Ð 104 (2) Ð 2 (0) 53 (2) 45 (0) 32 (1) 2 (0) 35 (0) 6 (0) 1 (1) 3 (0) 284 (6) 2.1

Ð 2 (0) 74 (21) Ð 3 (1) 3 (0) 4 (0) 5 (0) 4 (0) 2 (0) Ð Ð 1 (0) 98 (22) 22.4

Ð 1 (0) 81 (10) 1 (0) 2 (0) 62 (2) 42 (0) 20 (2) 1 (0) 27 (0) Ð Ð Ð 237 (14) 5.9

Total

% positive

5 (0)a 3 (0) 550 (53) 1 (0) 16 (1) 289 (20) 140 (1) 85 (9) 13 (2) 87 (1) 16 (1) 1 (1) 6 (0) 1,212 (89)

0 0 9.6 0 6.3 6.9 0.7 10.6 25.0 1.1 6.3 100 0 7.3

Number of pools tested (number of PCR pools positive); Þve mosquitoes per pool.

thoraces. Of the three Cx. erraticus individuals positive for D. immitis, two individuals had infections in their abdomens and the other individual was infected in the head or thorax. Discussion Ae. vexans was the dominant mosquito species found in this study and was most abundant April through early June. This species is associated with roadside ditches in northeast AR (Jamieson et al. 1994) and intermittent ßoodwaters (Gillies et al. 1971) along the margins of wetlands containing reeds, and is more abundant in spring, when water levels are at their highest (Sharkey et al. 1988). Another species, An. quadrimaculatus, became dominant June through August. To understand the seasonal dynamics of these two species, a thorough understanding of the landscape and agricultural practices is needed. One habitat known to be an important oviposition site for mosquitoes is rice Þelds, with rice acreage having a positive correlation with mosquito abundance (Washino 1980). In 2010, AR ranked Þrst in rice production in the United States (U.S. Department of Agriculture [USDA] 2012), with 1.79 million acres of rice planted (Child 2012). Rice Þelds extending into the edges of

towns (Quarterman et al. 1955), including Jonesboro, allow for signiÞcant mosquito nuisance issues. Stark and Meisch (1984) examined the distribution of mosquito species in rice Þelds in southeast AR. They found Ps. columbiae and An. quadrimaculatus to be most abundant when water levels were maintained on the Þelds (usually from late June until August or September) (Stark and Meisch 1984). They also found few Ae. vexans from June to August. In comparing the seasonal distribution of mosquitoes in our study with the studies of Stark and Meisch (1984), similar seasonal dynamics in the populations of Ae. vexans, An. quadrimaculatus, and Ps. columbiae were noted. To be an important mosquito species in vectoring heartworm, the infective stage (L3) needs to be conÞrmed, ensuring that D. immitis can fully develop (Ledesma and Harrington 2011). In this study, both Ae. vexans and Cx. p. quinquefasciatus had more pools positive in the head or thorax, indicating the presence of L3, infective larvae. These two species can therefore be considered important vectors in northeast AR. However, more studies are needed to determine the vector competence of the local strains of An. quadrimaculatus, as more An. quadrimaculatus pools (75%) were D. immitis positive only in the abdominal region. In addition, for the mosquitoes collected from the

Table 4. Number of mosquito pools tested and number of PCR pools positive for D. immitis in the Jonesboro, AR, area by month of collection Species Ae. vexans An. punctipennis An. quadrimaculatus Culex erraticus Cx. p. quinquefasciatus Culiseta inornata Psorophora columbiae Ps. ferox Ps. howardii Total % positive

2009 April

May

June

July

Aug.

Sept.

Oct.

120 (28) 5 (1) 5 (0) 2 (0) 1 (1) Ñ Ñ Ñ Ñ 133 (30) 22.5

108 (4) 6 (0) 4 (0) 5 (0) 29 (2) Ñ 12 (0) 4 (0) Ñ 168 (6) 3.6

95 (7) 3 (0) 36 (4) 9 (0) 8 (1) 1 (0) 17 (0) 1 (0) Ñ 170 (12) 7.1

77 (1) Ñ 94 (5) 32 (0) 6 (0) Ñ 52 (1) 1 (0) Ñ 262 (7) 2.7

51 (5) Ñ 101 (9) 65 (0) 13 (4) 1 (0) 6 (0) 5 (0) 1 (1) 243 (19) 7.8

47 (3) 1 (0) 38 (1) 26 (1) 9 (0) 1 (0) Ñ 3 (0) Ñ 125 (5) 4.0

52 (5) 1 (0) 11 (1) 1 (0) 19 (1) 10 (2) Ñ 2 (1) Ñ 96 (10) 10.4

876


Vol. 50, no. 4

Table 5. Number and percentage of mosquitoes positive for D. immitis from Caldwell, AR, collected from the kennel of a heartworm-positive dog Mosquito species Date

An. quadrimaculatus

Cx. erraticus

Ae. vexans

No. D. immitis positive/total no. analyzed 11 May 2009 8 July 2009 9 July 2009 10 July 2009 13 July 2009 14 July 2009 15 July 2009 Total Total % positive

Ñ 15/20 7/9 17/18 12/13 13/17 17/20 81/97 83.5

Ñ 0/1 0/1 1/1 Ñ 0/2 2/2 3/7 42.9

0/1 0/1 Ñ Ñ Ñ Ñ Ñ 0/2 0

kennel of an infected dog, 54.3% of individuals had L3 larvae associated in the head. Although An. quadrimaculatus has been found to be an important vector in other studies (Sauerman and Nayar 1983, Watts et al. 2001), our results give question to the ability of An. quadrimaculatus to transmit D. immitis in AR. Bradley and Nayar (1987) examined the Malpighian tubules of An. quadrimaculatus that were infected with D. immitis and found high mosquito mortality when the infective stage (L3) ruptured the tubules to begin their migration to the head region of the mosquito. We can assume this species feeds on microÞlaremic hosts in the wild (Ledesma and Harrington 2011), but more studies are needed on local strains of An. quadrimaculatus in AR to determine vector competence. Another species, Ps. columbiae, should be considered a weak vector of D. immitis in AR. Although it was fourth in abundance in our study, with 87 pools analyzed for D. immitis, only one pool was positive with L3 larva(e). We can assume the individual Ps. columbiae in this pool fed on microÞlaremic hosts in the wild, and these mosquitoes could potentially transmit heartworm. However, few pools were positive, leading us to question this speciesÕ ability to successfully develop L3 infective larvae. These are similar results to Jones and Meisch (1993), who collected mosquitoes within the city limits of Stuttgart, AR. They dissected 1,060 Ps. columbiae females and did not Þnd D. immitis in any of their specimens. Watts et al. (2001) also found few Ps. columbiae to be infected with D. immitis, even though it was fairly prevalent in Baton Rouge, LA. Ae. vexans can be considered an important vector for D. immitis, with 9.6% of pools for this species testing positive. Most of those positive pools were associated in early spring (Table 4). This provides information to pet owners in northeast AR that their pets may likely be more prone to acquiring infections of D. immitis in early spring, when Ae. vexans populations are at their highest. However, pet owners should take precaution in minimizing mosquito exposure time throughout the year, as there was considerable variation in the prevalence of D. immitis-infected mosquitoes among the six location sites (2.1Ð22.4%) and throughout the season (2.7Ð22.5%).

Mosquitoes

Ps. columbiae

Ñ 0/2 0/3 0/1 Ñ 0/1 0/1 0/8 0

Total D. immitis positive

Total analyzed

% positive D. immitis

0 15 7 18 12 13 19 84

1 24 13 20 13 20 23 114

0 66.7 53.8 90.0 92.3 65.0 82.6 73.7

Another aspect to determining an animalÕs exposure risk to heartworm infection is knowing if there are positive heartworm animals in the area. In our study, the frequency of D. immitis-infected mosquitoes collected near a heartworm-positive dog was much higher (73.7%) than in the original six collection sites (7.3%). Although mosquitoes were collected only from one positive dog, this study suggests that a single heartworm-positive dog potentially could increase infection pressure on susceptible animals sharing mosquito exposure. The percentages of positive mosquitoes inside the kennel of an infected dog ranged from 53.8 to 92.3%. These percentages are extremely high, and should bring awareness to owners and facilities with multiple dogs (i.e., shelters, training facilities). If a heartworm-positive dog is on the premises and exposed to mosquitoes, the other dogs in the kennel should be immediately removed and protected from being bitten by mosquitoes that may have had a bloodmeal from the positive dog. Owing to the use of UV light traps, the majority of mosquitoes collected were dusk- and night-time feeders. The population size of Aedes albopictus (Skuse) and other mosquitoes, such as Aedes triseriatus (Say), was likely underestimated owing to our surveillance method and may have been more commonly collected if Centers for Disease Control and Prevention gravid traps were used. Ae. albopictus, a day-time feeder (Robertson and Hu 1935), has been known to harbor D. immitis in the United States (Comiskey and Wesson 1995, Nayar and Knight 1999, Licitra et al. 2010). Another important mosquito species that was not collected in our study was Aedes japonicus (Theobald), formerly Ochlerotatus japonicus. Ae. japonicus was recently collected in northeast AR, including north of the city limits of Jonesboro (Gaspar et al. 2012), near location E in our study. This species is a day-time feeder (Tanaka et al. 1979), and adults can be collected using gravid traps (Caldwell et al. 2005, Thielman and Hunter 2006, Grim et al. 2007). Another study focusing on trapping day-time-feeding mosquitoes and their association with D. immitis is needed to access their potential as vectors for heartworm in AR. This is the Þrst study in North America to examine the potential for mosquitoes to harbor D. repens. This

July 2013

MCKAY ET AL.: PREVALENCE OF D. immitis IN MOSQUITOES

nematode is the causative agent for subcutaneous or ocular diroÞlariosis in the Old World (Simo´ n et al. 2009). Although no mosquitoes were positive for D. repens in our study, researchers in North America examining the vectors of Dirofilaria spp. using molecular techniques should consider D. repens in their analysis. Primers for D. repens are available (Rishniw et al. 2006). With the increase of international travel and the increase of unvaccinated puppies being imported into the United States (McQuiston et al. 2008), there is the enhanced risk of importing parasite species that are not endemic (Daugschies 2001). Rishniw et al. (2006) reported the Þrst case of D. repens infection in a 2-yr-old Labrador Retriever that was born in the Czech Republic and later imported into the Netherlands, Canada, and eventually into New York State. Mosquito species such as Ae. albopictus and Culex pipiens L. are known to vector D. repens in Italy (Cancrini et al. 2007). These mosquito species have known distributions in North America (Darsie and Ward 2005) and were present in our study. As D. repens is considered an emerging zoonotic disease in many parts of the Old World (Pampiglione and Rivasi 2000, AbdelÐRahman et al. 2008), it is a matter of time before another incident of D. repens is discovered in North America. The current study illustrates the importance of mosquitoes in the potential transmission of heartworm infection in northeast AR. It also demonstrates that there are speciÞc gaps in knowledge related to mosquitoÐ heartworm interactions. Future studies investigating the associations of mosquitoes in the vicinity of heartworm-positive animals, as well as examining day-time-feeding mosquitoes and the transmission efÞcacy of An. quadrimaculatus, would be beneÞcial. Acknowledgments We thank the Burns, Grippo and Rhodes families, T. Teague, C. Akin, J. Huff, and the Jonesboro Animal Control Shelter for access to their properties for mosquito collections. Further thanks to J. Gaspar, J. Owens, Z. Teague, M. Tisdale, and K. Quartermass for assistance in the Þeld and laboratory. We also appreciate B. Blagburn (Auburn University) for providing D. immitis-positive mosquitoes and C. Bourguinat (McGill University) for DNA sequence analysis. This research was supported by Novartis Animal Health U.S., Inc., Arkansas State University, AR Agricultural Experiment Station, and the Arkansas Biosciences Institute.

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