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Angiostrongylus vasorum and Eucoleus aerophilus in foxes (Vulpes vulpes) in Great Britain
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Veterinary Parasitology 154 (2008) 48–57 www.elsevier.com/locate/vetpar
Angiostrongylus vasorum and Eucoleus aerophilus in foxes (Vulpes vulpes) in Great Britain E.R. Morgan a,*, A. Tomlinson b, S. Hunter b, T. Nichols a,c, E. Roberts c, M.T. Fox c, M.A. Taylor b,c a
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK b Central Science Laboratory, Sand Hutton, York, UK c Royal Veterinary College, London, UK
Received 20 November 2007; received in revised form 19 February 2008; accepted 25 February 2008
Abstract The nematode parasite Angiostrongylus vasorum is a source of increasing concern in several parts of the world, where it causes significant disease in dogs. Wild canids, especially foxes, are likely to have a role in the epidemiology of canine infection, and the parasite could also affect fox health and population dynamics. The heart and pulmonary vasculature of 546 foxes culled mostly by gamekeepers in Great Britain in 2005–2006 were examined by dissection and a modified flushing technique. Forty foxes were found to be infected, giving an overall prevalence in the UK fox population of 7.3% (5.3–9.9). Prevalence varied widely between regions, from 0% (0–3) in Scotland and northern England to 23% (16–32) in south-east England. This closely matches the perceived incidence of disease in dogs, which is commonly diagnosed in the south-east but rarely in the north. In the Midlands, where disease has recently appeared in dogs, prevalence in foxes was 4.8% (2–11). Close geographical overlap of parasite distribution in foxes and dogs does not necessarily indicate an important wildlife reservoir of infection, but does suggest that A. vasorum might be spreading northwards. The hearts of infected foxes had thicker right ventricles than those of uninfected foxes, suggesting that the parasite could affect fox health and fitness. Burdens ranged from 1 to 59 adult nematodes. Sex, age and body condition were not significantly associated with infection. Eucoleus aerophilus and Crenosoma vulpis, nematode parasites of the respiratory system, were found in 213 and 11 foxes respectively, with slightly higher prevalence of E. aerophilus in the south and east. No specimens of the heartworm Dirofilaria immitis were found, giving an upper 95% confidence interval for prevalence of 0.84%. # 2008 Elsevier B.V. All rights reserved. Keywords: Parasite; Nematode; Cardiopulmonary; Lungworm; Capillaria; Crenosoma; Angiostrongylosis; Wildlife disease; Reservoir; Epidemiology
1. Introduction The nematode Angiostrongylus vasorum (Baillet, 1866) is parasitic in the right ventricle and pulmonary arteries of the red fox (Vulpes vulpes) and dog (Canis familiaris) (Bolt et al., 1994), and also infects the wolf
* Corresponding author. Tel.: +44 117 9287485. E-mail address: [email protected] (E.R. Morgan). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.02.030
(Canis lupus) (Segovia et al., 2001), coyote (Canis latrans) (Bourque et al., 2005) and other canid, mustelid and even felid species (Jeffery et al., 2004). Its distribution is worldwide but patchy, with ‘hyperendemic’ areas of high prevalence surrounded by regions in which the parasite is rarely found. In recent years, A. vasorum has increasingly been recorded outside these known hyper-endemic foci (Morgan et al., 2005). This is of great concern to veterinarians and dog owners, since infection with A. vasorum can cause
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severe disease in dogs (Chapman et al., 2004; Koch and Willesen, in press). It is widely assumed that fox populations act as a reservoir of infection for dogs (Bolt et al., 1992), although the relative contribution of larvae shed by dogs and by foxes to canine infection has yet to be determined. Pathological changes have been observed in infected foxes (Poli et al., 1984, 1991; Simpson, 1996), but the effect of A. vasorum on vulpine health and population dynamics similarly remains unknown. In the UK, autocthonous infection of dogs with A. vasorum was first recorded in Cornwall (Simpson and Neal, 1982), with further cases appearing in south Wales (Patteson et al., 1987; Trees, 1987). The parasite was considered to be largely confined to these areas (Patteson et al., 1993; Martin et al., 1993) until increasing numbers of cases in south-east England confirmed significant spread to this area (Kriek, 2001; Chapman et al., 2004). Recent anecdotal reports of canine disease suggest that A. vasorum is expanding its range northwards. However, such reports are unreliable indicators of parasite distribution since parasitological diagnosis is rarely confirmed, and movement of dogs often makes it impossible to define exactly where they were infected. Foxes infected with A. vasorum were found close to the focus of canine disease in Cornwall (Simpson, 1996), but systematic data on distribution and levels of infection in foxes in the UK are absent. The present study aimed to determine the extent to which A. vasorum has expanded its range outside existing hyper-endemic areas of Great Britain, so that the risks of canine infection can be geographically defined. Associations between infection and various host factors were also examined in order to advance understanding of parasite epidemiology. Similar questions were addressed in relation to the nematode Eucoleus aerophilus (syn. Capillaria aerophila), which is parasitic in the respiratory tract and was coincidentally recovered, along with Crenosoma vulpis, when dissecting lungs for A. vasorum. 2. Materials and methods 2.1. Study animals Hearts and lungs were obtained from a total of 546 foxes (V. vulpes) culled as part of pest control programmes in different parts of Great Britain from January 2005 to April 2006. Sampling was opportunistic and some regions, including south Wales and Cornwall, and urban foxes, were under-represented. The foxes were shot and the carcases collected within
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1–2 days. Foxes were classified by sex, age (young if incisors clean and unworn; adult if incisors showed loss of lobation and brown dentin spots on occlusal surfaces) and body condition (good, fair or poor, on the basis of lumbar and retroperitoneal fat thickness). The heart and lungs were removed together, taking care not to rupture the pulmonary trunk, and frozen at 20 8C. 2.2. Parasite recovery (heart) Hearts and lungs were dissected over the next 6–15 months, and extraction of A. vasorum was prioritised over that of other species. After exteriorisation from the pericardium, both ventricles were incised transversely mid way between the base and the apex of the heart. In a sub-set of hearts, the maximum thickness of the outer wall of each ventricle at this point was measured using callipers. The heart was separated from the lungs by transecting the major vessels as close as possible to the lungs, and all chambers as well as the pulmonary arterial trunk opened and inspected visually for nematodes. Blood clots were broken up by digital pressure and the heart chambers rinsed out over a tray, as well as the plastic bag in which the organs had been contained. The washings were inspected visually in the tray before being passed through sieves of 150 and 38 mm aperture. The residue on each sieve was resuspended in water and examined for nematodes under a dissecting microscope. The trachea was opened longitudinally and inspected closely for nematodes, which were removed, and a scalpel scraping of the mucosal surface was smeared onto a slide and examined under a transmission microscope at total magnification 100 for nematode eggs and larvae. 2.3. Parasite recovery (lungs) The lung lobes were separated into left and right groups. One side was selected at random for further dissection, and the other for flushing. Dissection consisted of opening all visible pulmonary vessels down to the narrowest practicable point (about 0.5 mm diameter) and picking out any nematodes seen. Thereafter, bronchi were likewise dissected and then rinsed with water over a sieve of 38 mm aperture, with the washings examined under the dissecting microscope. Lobes designated for flushing were separated into cranial and caudal groups and a modification of Jeffery’s (2004) technique applied. The largest blood vessel in each was identified and a pipette tip inserted firmly into the lumen and attached to a rubber hose. Water was pumped through using tap pressure and
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collected as it oozed out of the lungs and airways. Flushing continued until the lungs appeared pale and devoid of blood and the fluid appeared clear. If any parts of the lung appeared under-perfused the pipette tip was moved to a new vessel and flushing continued. The washings were sieved and examined as before. After flushing, the blood vessels and airways were dissected to their smallest practicable diameter and any nematodes picked out. No difference was found in the rate of recovery of Angiostrongylus using dissection or flushing followed by dissection after the first 200 foxes, but Eucoleus and Crenosoma were more often recovered from the flushed lobes. Subsequent lungs were therefore processed by flushing followed by dissection of all lobes. Nematodes were mounted on a microscope slide in water and identified under the transmission microscope, before being frozen in normal saline. The three nematode species known to occur in the cardiopulmonary tract of foxes in the UK – A. vasorum, C. vulpis and E. aerophilus (syn. C. aerophila) – are easily distinguishable on basic morphology (Taylor et al., 2007). 2.4. Statistical analyses Associations between the presence of nematodes and fox sex, age, body condition, geographical location and season were assessed separately for A. vasorum and E. aerophilus using logistic regression. Association between burdens of each species was estimated by Spearman rank correlation. Regions were defined by dividing Ordnance Survey National Grid references into arbitrary categories representing climatically different parts of the country. The prevalence of parasitism was compared across regions and seasons using the Chisquare test for association, and parasite abundance and intensity of infection using Kruskal–Wallis tests on raw data and one-way ANOVA on log10 (x + 1) transformed data (Ro´zsa et al., 2000). Observer bias was examined by Chi-square comparison of observed and expected frequency of parasite occurrence. 95% Confidence
intervals for prevalence were estimated using the exact binomial method. The geographical distribution of A. vasorum was assessed for clustering using Kulldorff’s spatial scan statistic, within a purely spatial Bernoulli model. Spatial autocorrelation was assessed by Moran’s I statistic, with adjacency defined as shared boundaries at the county level. Statistical analyses were conducted using SPSS 12.0 (SPSS Inc., Chicago), ArcGIS 9.x (ESRI, California), SatScan v7.0.3 (SaTScanTM, Boston) and GeoDa version 0.9.5i (Spatial Analysis Lab, Illinois) computer packages. 3. Results 3.1. Parasite identification and overall prevalence Nematodes were found in 44% (95% confidence intervals, CI 39.7–48.1) of foxes. E. aerophilus was by far the most common species, followed by A. vasorum (Table 1). Too few C. vulpis were found to justify inclusion of this species in further analyses. Body condition was also excluded given that there were no animals in poor condition and only 23 in fair condition, and preliminary analysis showed that condition was not significantly associated with prevalence of any nematode species. Logistic regression revealed that A. vasorum was highly significantly over-represented in the south-east (Table 2). A. vasorum was equally common in both sexes, but E. aerophilus was more common in males than in females (prevalence = 0.61, CI 0.52–0.69 in males and 0.41, CI 0.32–0.50 in females). E. aerophilus prevalence also varied by region, being more common in the southern and eastern regions. Infection was not significantly associated with age category for either nematode species. 3.2. Anatomical location and extraction Only 41% of the A. vasorum recovered were found in the heart and pulmonary trunk artery, the rest being recovered from the smaller vessels by flushing and fine
Table 1 Nematodes found in the cardiopulmonary tract of 546 foxes Species
Angiostrongylus vasorum Crenosoma vulpis Dirofilaria immitis Eucoleus aerophilus
Prevalence
0.07 0.02 0 0.39
CI = exact binomial 95% confidence interval.
CI
0.05–0.10 0.01–0.03 0.00–0.01 0.35–0.44
Intensity Mean
Variance
Range
6.7 5.6 – 4.4
88.1 54.0 – 40.5
1–59 1–22 – 1–51
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Table 2 Composition of the binary logistic regression model and significant predictors for nematode infection Variable
d.f.
Category
n
(a) Model constituents Region
4
North Midlands South East South-East
138 126 29 128 125
Season
3
Winter (December to February) Spring (March to May) Summer (June to August) Autumn (September to November)
120 190 90 146
Sex
2
Not known Male Female
275 143 128
Age
2
Not known Young (<1 year) Adult (>1 year)
230 265 51
Factor
d.f.
Wald statistic
p
Significant levels
(b) Significant predictors of A. vasorum infection. HR = 2.951, 8 d.f., p = 0.937 Region 4 27.120 <0.001 South-East
p
OR
CI
<0.001
6.75
2.51–18.2
Summer Autumn
0.019 0.029
5.00 3.45
1.31–19.2 1.14–10.5
(c) Significant predictors of E. aerophilus infection. HR = 0.624, 8 d.f., p = 0.624. Region 4 11.101 0.025 South East
0.031 0.002
2.63 2.40
1.09–6.33 1.38–4.18
0.001
2.44
1.47–4.06
Season
Sex
3
2
8.551
29.830
0.036
<0.001
Male
HR = Hosmer–Lemeshow goodness of fit Chi-square statistic, d.f. = degrees of freedom, OR = odds ratio and CI = 95% confidence intervals.
dissection. Of foxes ultimately determined to be positive for A. vasorum, only 36% would have been detected by dissection of the heart and pulmonary trunk alone. Larvae were found on the tracheal scrape in only 10% of infected animals. There was no correlation between parasite burden and the proportion of A. vasorum found in the smaller pulmonary vasculature, and no significant difference in the number recovered from the lungs by dissection and flushing. In several cases nematodes were dissected from the airways after flushing had been completed. Of animals positive for E. aerophilus, 55% were detected by gross dissection of the trachea and bronchi (of which 62% were also positive on flushing), and a further 34% by recovery of adults or fragments of adults in the lung flush, with the remainder detected only by observation of eggs on the tracheal scrape. However, eggs were detected in the trachea of only 26% of foxes that carried adult E. aerophilus, even though around half of the infrapopulations included gravid female worms. Just a single adult worm was found in 37% of animals positive for E. aerophilus, and two worms in 18%. For A.
vasorum, 18% of infrapopulations comprised a single adult or immature worm. 3.3. Geographical distribution The broad-scale geographical distribution of the two most common nematode species is summarised in Table 3. Although very few C. vulpis were recovered, this species was found in three of the five regions, and 6 of the 11 infected foxes were from the North. E. aerophilus was fairly evenly distributed between regions, but the occurrence of A. vasorum was more varied (Fig. 1). Most of the infected foxes were found in south-east England, and groups of infected foxes in the south, east and Midlands were interspersed by areas of low prevalence. No A. vasorum were recovered from foxes in the far north of England or in Scotland. Spatial analysis pointed to a significant cluster of infection in the south-east, centred on National Grid Reference SU 200500 and with a radius of 84.4 km. Within this area 38 foxes were sampled, of which 20 were infected compared with an expected
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Table 3 Regional variation in the prevalence of cardiopulmonary nematodes of foxes in Great Britain Region
North Midlands South East South-east
OS National grid squares
NJ-NY, SD-SJ SK, SO, SP ST, SY TA-TM SU, SZ, TQ-TV
Total
A. vasorum
E. aerophilus
Prevalence (%)
CI
Prevalence
CI
0 4.8 6.9 1.6 23.2
0–3.0 2.0–11 1.2–24 0.2–6.2 16–31
40 39 55 45 30
32–49 30–49 36–73 37–54 23–39
7.1
5.2–9.7
40
36–44
OS = ordnance survey. See Table 2 for sample sizes.
number of 3 (relative risk 14.044, log likelihood ratio 33.1, p < 0.001). Spatial autocorrelation in prevalence between counties was of borderline significance (Moran’s I = 0.0694, p = 0.086) (Fig. 2).
Fig. 1. The geographical distribution of Angiostrongylus vasorum in foxes in Great Britain. Clear circles represent foxes clear of infection, and filled squares infected individuals. Some markers overlie each other where foxes were collected from the same location. The figure illustrates the relative distribution of sampling effort and positive samples. See text for formal spatial analysis.
Fig. 2. Prevalence of A. vasorum by county. Counties not sampled are not distinguished from those with zero prevalence (see Fig. 1). The sampling design was non-probabilistic and prevalence is indicative only, see text for formal comparison of regional occurrence.
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Patterns in prevalence were generally echoed by parasite abundance and to a lesser extent by the intensity of infection. All species were overdispersed in distribution among hosts, with variance in abundance greatly exceeding the mean (Table 1). A. vasorum abundance differed significantly between regions (Kruskall–Wallis x2 = 66.7, 4 d.f., p < 0.001; ANOVA F4, 541 = 16.876, p < 0.001), and was higher in the south-east than in all other regions (Tukey’s pairwise comparisons p 0.004). Intensity of infection was also higher in the south-east (mean 7.6, variance 113.7) than in other regions (range 0– 5.0), but the difference was not significant. 3.4. Seasonal patterns A. vasorum was more commonly found in foxes killed in summer and autumn (Table 2), but there were no significant seasonal differences in abundance. E. aerophilus prevalence did not vary by season. 3.5. Parasite associations There was no association between infection with E. aerophilus and A. vasorum (x2 = 0.051, 1 d.f., p > 0.50), and no correlation between the burden of each species in individual foxes (Spearman r = 0.02, p = 0.58). 3.6. Cardiopulmonary pathology Although the mean thickness of the left ventricle was not significantly different between infected and noninfected groups (overall mean 17.48 mm S.E. 0.31, n = 34), the right ventricle was significantly thicker in infected foxes (infected 7.1 mm S.E. 0.22, n = 17; uninfected 5.0 0.16, n = 17; mean difference 2.1 0.27, t = 7.71, 32 d.f., p < 0.001). The ratio of right to left ventricular thickness was also greater in infected than in uninfected foxes (0.43 0.02 cf. 0.28 0.01, t = 8.97, 32 d.f., p < 0.001). Histological examination of lung tissue from foxes infected (n = 10) and uninfected (n = 10) with A. vasorum suggested that parasitism was associated with breakdown in alveolar structure (honeycomb lung) and inflammatory cell infiltration. However, lung structure was also disrupted in uninfected foxes, presumably as a result of the repeated freezing and thawing of hearts and associated autolysis, so observed changes are not described further. 3.7. Observer bias Eight identically trained observers dissected the hearts and lungs, each contributing between 22 and 95
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samples to the total. The frequency with which each observer found nematodes did not differ significantly from that expected by chance for A. vasorum (x2 = 7.119, 7 d.f., p > 0.10), but there was significant observer bias for E. aerophilus (x2 = 42.84, 7 d.f., p < 0.001). Within-observer prevalence for this species varied from 13 to 61%, and two observers detected E. aerophilus more often than expected. Since samples were drawn fairly randomly from the stored material, this did not lead to bias in regional prevalence or other risk factors for infection, so data were analysed in full without adjustment. A sub-sample of 137 negative hearts and lungs were frozen after dissection and thoroughly re-examined by different observers several months later. Four of these samples contained 1 or 2 adult A. vasorum that had been previously undetected. 4. Discussion Since two of the three known endemic areas for A. vasorum in the UK (Cornwall and south-west Wales) were not sampled, prior expectation in the present study was to find this species only in south-east England. In fact, low levels of infection were also discovered in foxes in other parts of southern and eastern England and the Midlands. The lack of previous studies on this scale makes it impossible to demonstrate that the parasite is spreading northwards, although our data and the expanding range from which canine cases are reported are consistent with this hypothesis. Whether foxes act as the main reservoir of infection for dogs or not, veterinarians should be aware of the possibility of canine angiostrongylosis outside areas in which it has previously been diagnosed, since the parasite is present and transmission is clearly possible across a large part of the UK. Although the foxes sampled here were mostly rural and culled by gamekeepers, urban and sub-urban foxes presumably have greater opportunities for contact with dogs and could be more important in terms of disease transmission across the wildlife-domestic animal boundary. This appears to be the case for the better studied cestode parasite Echinococcus multilocularis (Deplazes et al., 2004; Morgan et al., 2004). In Denmark, the highest prevalence of A. vasorum was found in urban and suburban foxes in Copenhagen (Saeed et al., 2006). Future studies should aim to sample urban as well as rural foxes for parasites, for example, through examination of road-killed animals or fresh faeces. A. vasorum was not found in foxes north of the Midlands, and although the possibility of foci of infection above this latitude cannot be excluded, prevalence is clearly much lower in the north than
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the south of Great Britain. However, it is not clear whether northern areas are relatively unsuitable for transmission, or whether they could sustain a parasite population but have yet to be colonised. Given the frequent long-range movement of dogs within the UK, if conditions exist to support the parasite it can only be a matter of time before it becomes established in all suitable regions. The introduction of infection by dogs and establishment in local fox populations is considered to underlie the spread of A. vasorum in Scandinavia ˚ blad et al., 2003; Saeed et al., 2006). Unfortunately, (A too little is known about the parasite’s climatic and biological requirements to make sensible predictions about areas at risk from range expansion. The intermediate host range is broad (Guilhon and Cens, 1973) and northern parts of Great Britain certainly harbour competent slug and snail vectors. C. vulpis, which also uses gastropod mollusc intermediate hosts, was found in several foxes in the Scottish borders. On the Canadian island of Newfoundland, C. vulpis is widely distributed in foxes, but A. vasorum inhabits only areas with a mean winter temperature above 4 8C (Jeffery et al., 2004). Since this applies to the whole of Great Britain, it seems likely that there is no absolute climatic obstacle to the establishment of either species, although higher gastropod densities in mild and humid parts of the country may favour transmission (Simpson and Neal, 1982). A. vasorum has been found in foxes in a wide range of countries with differing climates (Bolt et al., 1994), and the reasons for its apparently patchy distribution in the UK and elsewhere are unknown. In this context, the marginal level of autocorrelation in A. vasorum prevalence found at county level in this study, along with the single identifiable cluster of infection in the south-east, is interesting. If spread were by means of organic expansion of established hyper-endemic foci, stronger autocorrelation would be expected, at least until foci begin to coalesce. Our results might indicate that the focal geographical distribution of A. vasorum in the UK has already broken down into a more generalised colonisation of the southern half of the country. However, the arbitrary nature of the county boundaries, the limited sample size, and especially the omission of foxes from south-west Wales and Cornwall, limit the strength of the conclusions that can be drawn. Ultimately, tracking parasite spread will require longitudinal studies. In Denmark, the abundance of E. aerophilus, but not A. vasorum, was found to correlate with fox population density (Saeed et al., 2006). The lack of detailed estimates of fox density in the UK and most other countries (Webbon et al., 2004) limits the predictive
value of this parameter, while threshold host densities for A. vasorum persistence are not known. The emergence of A. vasorum in southern England in the 1990s coincided with an outbreak of sarcoptic mange that reduced fox populations locally by as much as 90% (Baker et al., 2000; Simpson, 2002), although in some areas urban fox populations appeared to increase during this period (Wilkinson and Smith, 2001). The importance of fox population density in enabling the parasite to become established is therefore unclear, while other effects such as increased dispersal (Baker et al., 2000) and possibly immuno-suppression could exacerbate parasite spread during mange outbreaks even as overall population density decreases. It is perhaps worth noting that several European countries have experienced outbreaks of sarcoptic mange in foxes in recent years (Simpson, 2003), including Spain and Denmark, which also have locally high prevalence of A. vasorum (Willingham et al., 1996; Segovia et al., 2004). Sarcoptic mange has been found to be associated with the presence of helminths in foxes in Italy (Balestrieri et al., 2006). It is likely that interactions between climate and intermediate and definitive host densities influence the chances of parasite establishment and persistence in new areas. In this context, climate change and associated alterations in slug and snail phenology (Root et al., 2003) could affect the global distribution of A. vasorum and the local risk of infection for foxes and dogs. E. aerophilus is clearly common in foxes in all parts of Great Britain. Regional differences appear to exist, but are smaller in magnitude than observer variation and must be considered slight at most. This apparent stability in level of infection is also a feature elsewhere in Europe, where prevalence in foxes is typically between 35 and 65% (Manˇas et al., 2005). The parasite appears to be more common in Denmark (74% prevalence; Saeed et al., 2006) and Norway (88%; Davidson et al., 2006), although recovery methods were different. In the present study, no relationship was found between season and risk of infection. This could be explained by lack of seasonality in transmission, parasite longevity that exceeds the timescale of seasonal effects, or damping of seasonal effects, for example by density dependence or immunity. E. aerophilus was found more often in male than in female foxes, in line with theories of immune handicap in male mammals (Wilson et al., 2002). Although no relationship was found between E. aerophilus and A. vasorum infection, other workers have recorded a positive correlation in burdens of these species, and a negative correlation between A. vasorum and C. vulpis, which they took to
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indicate that different intermediate host species are used (Saeed et al., 2006). Jeffery et al. (2004), in contrast, found no relationship between burdens of A. vasorum and C. vulpis in individual foxes. C. vulpis was rarely found in the present study, but this is not considered an accurate estimate of prevalence since the small size of the nematode and observer prioritisation of the larger species are likely to have decreased detection rate. Sensitivity of observed prevalence to method of dissection and observer experience might explain some of the wide variation in prevalence (5–75%) previously reported for C. vulpis in foxes in other parts of Europe (Davidson et al., 2006). The impact of cardiopulmonary nematode infection on fox health and population dynamics is unknown. C. vulpis and A. vasorum are well recognised causes of disease in domestic dogs (Martin et al., 1993; Shaw et al., 1996; Chapman et al., 2004), and have been associated with pulmonary pathology in foxes (Poli et al., 1991; Conboy and Adams, 1995). E. aerophilus has been found in a wide range of carnivore species including humans, sometimes in association with pathological changes in the lungs and airways (e.g. Crum et al., 1978; Morrison and Gier, 1978; Greenlee and Noone, 1984; Laux, 1987; Richardson et al., 1992; Skirnisson et al., 1993; Martin, 1998; Nevarez et al., 2005; Nelson et al., 2007; Krone et al., 2008; Lalosevic et al., 2008). The poor condition of the tissue in this study made detailed histopathology of dubious worth, so changes are not described in detail. However, the right ventricle of the heart was significantly thickened in animals in which A. vasorum was found. Poli et al. (1984) also noted right ventricular hypertrophy in infected foxes, although Jeffery et al. (2004) failed to find this even at much higher burdens than we observed, and in fact infection was associated with reduced overall heart weight relative to body weight. Jeffery et al. (2004), like us, found no difference in body condition between infected and uninfected foxes. The implications of A. vasorum infection for vulpine health and fitness are uncertain and would be difficult to measure. Cross-sectional studies are a notoriously insensitive way of detecting effects of parasitism on wildlife populations (Irvine, 2006), but, in the absence of sustained funding for wildlife disease surveillance and interventional studies, often constitute the only available data. The present study found an increased prevalence of angiostrongylosis in summer and autumn. A lack of any seasonal effect in most other published surveys can be explained by concentrated collection of carcases during short hunting seasons, and consequently limited
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seasonal range. Given the long pre-patent period of A. vasorum, the observed pattern could reflect increased transmission in spring and summer. Foxes are likely to include more amphibians in their diets at this time of year, providing greater opportunity for infection by ingestion of paratenic hosts (Bolt et al., 1993, 1994). Season is confounded by age in foxes, since cubs are born in spring, but the observed seasonal pattern cannot be explained by age-dependent infection since there was no difference in A. vasorum prevalence or intensity between age categories. It is perhaps surprising that no age effect was found for either A. vasorum or E. aerophilus. This could be explained by different combinations of age-dependent acquisition, acquired immunity and parasite-induced host mortality. However, cross-sectional data, especially at such crude age resolution, constitute a blunt tool for the detection of such dynamics. It should also be borne in mind that the prevalence and intensity of infection with A. vasorum were lower in this study than in comparable surveys in Denmark (49%, mean intensity 7.4; Saeed et al., 2006) and Newfoundland, Canada (56%, 230; Jeffery et al., 2004), and host-parasite relationships could be radically different at higher levels of infection. Indeed, Jeffery et al. (2004) considered the lack of a positive age– intensity relationship to indicate that immunity was limiting burdens of A. vasorum in foxes in Newfoundland, while C. vulpis intensity actually decreased with age. At lower overall parasite abundance in Denmark, prevalence of A. vasorum (and also E. aerophilus) increased with age, although mean intensity did not change (Saeed et al., 2006). Higher intensity of infection could also affect anatomical location of the adult parasites: thus, Jeffery found that 78% of infected foxes had A. vasorum in the heart, compared with 36% in the present study, which might be explained by competitive displacement of worms from the arteries at high infrapopulation size (Sukhdeo and Bansemir, 1996). Flushing the lungs and then dissecting the pulmonary arteries was found to be an effective way of detecting A. vasorum, although it is difficult to concurrently maximise the detection rates of all species of cardiopulmonary nematode. Importantly, surveys of A. vasorum that are limited to dissection of the heart and major pulmonary vessels are likely to seriously underestimate both the prevalence and abundance of this species. The flushing technique causes physical disruption of the lung tissue and is less suitable for studies in which the anatomical position of the nematodes and the structure of the tissue are important. It is also time-consuming, taking experienced investigators around 1 h per animal. Given
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that coarse dissection of the heart and pulmonary trunk is some 10 times quicker, the reduced sensitivity of this method might be offset by increased sample size when investigator time rather than access to fox carcases is the major limiting factor, and the important variable is presence of the parasite rather than accurate estimates of prevalence or abundance. It is also notable that in the present study the main patterns in relation to geographical distribution, season and host sex were evident using prevalence data alone. Analysis of abundance and intensity generally reinforced these patterns but was less sensitive in detecting them. From our data, prevalence therefore appears to be the most useful single variable in detecting patterns in level of parasitism within populations, even though it is very sensitive to differences in detection efficiency between studies. The present study demonstrates that A. vasorum is more widespread in foxes in Great Britain than previously supposed, and suggests that northward spread is likely. Our data provide an important baseline for future studies. As well as surveying parasite distribution, future work should attempt to elucidate the factors important to parasite transmission and abundance so that the risks of range expansion can be more precisely assessed. Acknowledgements We are grateful for the assistance of Colin Morgan, Vicky Jackson and other CSL staff in organising the fox collection and sample storage, and of Nicholas Ilchyshyn, Tanya Kahrs, Peter Ward, Madeleine Krajewski, Hannah Ridout, William Low, Charlotte Young, Dhanurjaya Thyagaraja, Gemma Jones and especially Hannah Rose and Kira Sawyer in conducting the dissections. The Nuffield Trust and the Wellcome Trust provided financial support for summer studentships. Fox collections were facilitated as part of an ongoing project on trichinellosis funded by the UK Food Standards Agency (FSA). We thank Vic Simpson for his encouragement and useful discussions throughout this work. References ˚ blad, B., Christensson, D., Lind, E.O., Agren, E., Morner, T., 2003. A Angiostrongylus vasorum etablerad i Sverige. Sven. Veterina¨rtidning 12, 11–15 (In Swedish). Baker, P.J., Funk, S.M., Harris, S., White, P.C.L., 2000. Flexible spatial organization of urban foxes, Vulpes vulpes, before and during an outbreak of sarcoptic mange. Anim. Behav. 59, 127– 146.
Balestrieri, A., Remonti, L., Ferrari, A., Lo Valvo, T., Robetto, S., Orusa, R., 2006. Sarcoptic mange in wild carnivores and its cooccurrence with parasitic helminths in the Western Italian Alps. Eur. J. Wildl. Res. 52, 196–201. Bolt, G., Monrad, J., Henriksen, P., Dietz, H.H., Kock, J., Bindseil, E., Jensen, A.L., 1992. The fox (Vulpes vulpes) as a reservoir for canine angiostrongylosis in Denmark. Acta Vet. Scand. 33, 357–362. Bolt, G., Monrad, J., Frandsen, F., Henriksen, P., Dietz, H.H., 1993. The common frog (Rana temporaria) as a potential paratenic and intermediate host for Angiostrongylus vasorum. Parasitol. Res. 79, 428–430. Bolt, G., Monrad, J., Koch, J., Jensen, A.L., 1994. Canine angiostrongylosis: a review. Vet. Rec. 135, 447–452. Bourque, A., Whitney, H., Conboy, G., 2005. Angiostrongylus vasorum infection in a coyote (Canis latrans) from Newfoundland and Labrador, Canada. J. Wildl. Dis. 41, 816–819. Chapman, P.S., Boag, A.K., Guitan, J., Boswood, A., 2004. Angiostrongylus vasorum infection in 23 dogs. J. Small Anim. Pract. 45, 435–440. Conboy, G.A., Adams, C., 1995. Treatment of Crenosoma vulpis infection in two silver foxes (Vulpes vulpes) with ivermectin. J. Zoo Wildl. Med. 26, 597–600. Crum, J.M., Nettles, V.F., Davidson, W.R., 1978. Studies on endoparasites of black bear (Ursus americanus) in South-East United States. J. Wildl. Dis. 14, 178–186. Davidson, R.K., Gjerde, B., Vikøren, T., Lillehaug, A., Handeland, K., 2006. Prevalence of Trichinella larvae and extra-intestinal nematodes in Norwegian red foxes (Vulpes vulpes). Vet. Parasitol. 136, 307–316. Deplazes, P., Hegglin, D., Gloor, S., Romig, T., 2004. Wilderness in the city: the urbanization of Echinococcus multilocularis. Trends Parasitol. 20, 77–84. Greenlee, P.G., Noone, K.E., 1984. Pulmonary capillariasis in a dog. J. Am. Anim. Hosp. Assoc. 20, 983–985. Guilhon, J., Cens, B., 1973. Angiostrongylus vasorum (Baillet, 1866): e´tude biologique et morphologique. Ann. Parasitol. Hum. Comp. 48, 567–596. Irvine, R.J., 2006. Parasites and the dynamics of wild mammal populations. Anim. Sci. 6, 775–781. Jeffery, R.A., Lankester, M.W., McGrath, M.J., Whitney, H.G., 2004. Angiostrongylus vasorum and Crenosoma vulpis in red foxes (Vulpes vulpes) in Newfoundland, Canada. Can. J. Zool. 82, 66–74. Koch, J., Willesen, J.L., in press. Canine pulmonary angiostrongylosis: an update. Vet. J., doi:10.1016/j.tvjl.2007.11.014, available online 3rd January 2008. Kriek, J.G., 2001. Angiostrongylus vasorum in a dog in Surrey. Vet. Rec. 148, 56. Krone, O., Guminsky, O., Meinig, H., Herrman, M., Trinzen, M., Wibbelt, G., 2008. Endoparasite spectrum of wild cats Felis silvestris (Felis catus L.) from the Eifel, Pfalz region and Saarland, Germany. Eur. J. Wildl. Res. 54, 95–100. Lalosevic, D., Lalosevic, V., Klem, I., Stanojev-Jovanovic, D., Pozio, E., 2008. Pulmonary capillariasis miming bronchial carcinoma. Am. J. Trop. Med. Hyg. 78, 14–16. Laux, A., 1987. Extensity and intensity of the infection with endoparasites in hedgehogs. Angew. Parasitol. 28, 137–141. Manˇas, S., Ferrer, D., Castella`, J., Lo´pez-Martı´n, J.M., 2005. Cardiopulmonary helminth parasites of red foxes (Vulpes vulpes) in Catalonia, northeastern Spain. Vet. J. 169, 118–120. Martin, M., 1998. Lungworm infection in dogs. Vet. Rec. 143, 568. Martin, M.W.S., Ashton, G., Simpson, V.R., Neal, C., 1993. Angiostrongylosis in Cornwall: clinical presentations of eight cases. J. Small Anim. Pract. 34, 20–25.
E.R. Morgan et al. / Veterinary Parasitology 154 (2008) 48–57 Morgan, E.R., Milner-Gulland, E.J., Torgerson, P.R., Medley, G.F., 2004. Ruminating on complexity: macroparasites of wildlife and livestock. Trends Ecol. Evol. 19, 181–188. Morgan, E.R., Shaw, S.E., Brennan, S.F., De Waal, T.D., Jones, B.R., Mulcahy, G., 2005. Angiostrongylus vasorum: a real heartbreaker. Trends Parasitol. 21, 49–51. Morrison, E.E., Gier, H.T., 1978. Lungworm in coyotes on Great Plains. J. Wildl. Dis. 14, 314–316. Nelson, T.A., Gregory, D.G., Burroughs, C., Laursen, J.R., 2007. Prevalence of lungworm in Illinois coyotes. Trans. Illinois State Acad. Sci. 100, 89–95. Nevarez, A., Lopez, A., Conboy, G., Ireland, W., Sims, D., 2005. Distribution of Crenosoma vulpis and Eucoleus aerophilus in the lung of free-ranging red foxes (Vulpes vulpes). J. Vet. Diagn. Invest. 17, 486–489. Patteson, M.W., Wotton, P.R., Lucke, V.M., Wright, A.I., Gibbs, C., 1987. Angiostrongylus vasorum in a terrier. Vet. Rec. 120, 349. Patteson, M.W., Gibbs, C., Wotton, P.R., Day, M.J., 1993. Angiostrongylus vasorum in seven dogs. Vet. Rec. 133, 556–570. Poli, A., Arispici, A., Marconcini, A., Manciati, F., de Monte, D., 1984. Angiostrongylus vasorum (Baillet, 1866) in red foxes (Vulpes vulpes L.) in Italy. J. Wildl. Dis. 20, 345–346. Poli, A., Arispici, M., Mancianti, F., Abramo, F., 1991. Pathology of naturally acquired Angiostrongylus vasorum infection in the red fox (Vulpes vulpes). Angew. Parasitol. 32, 121–126. Richardson, D.J., Owen, W.B., Snyder, D.E., 1992. Helminth parasites of the raccoon (Procyonis lutor) from North-Central Arkansas. J. Parasitol. 78, 163–166. Root, T.L., Price, J.T., Hall, K.R., Schneider, S.H., Rosenzweig, C., Pounds, J.A., 2003. Fingerprints of global warming on wild animals and plants. Nature 421, 57–60. Ro´zsa, L., Reiczigel, J., Majoros, G., 2000. Quantifying parasites in samples of hosts. J. Parasitol. 86, 228–232. Saeed, I., Maddox-Hyttel, C., Monrad, J., Kapel, C.M.O., 2006. Helminths of red foxes (Vulpes vulpes) in Denmark. Vet. Parasitol. 139, 168–179. Segovia, J.M., Torres, J., Miquel, J., Llaneza, L., Feliu, C., 2001. Helminths in the wolf, Canis lupus, from north-western Spain. J. Helminthol. 75, 183–192.
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Segovia, J.M., Torres, J., Miquel, J., 2004. Helminth parasites of the red fox (Vulpes vulpes L. 1758) in the Iberian peninsula: an ecological study. Acta Parasitol. 49, 67–79. Shaw, D.H., Conboy, G.A., Hogan, P.M., Horney, B.S., 1996. Eosinophilic bronchitis caused by Crenosoma vulpis infection in dogs. Can. Vet. J. 37, 361–363. Simpson, V.R., 1996. Angiostrongylus vasorum infection in foxes (Vulpes vulpes) in Cornwall. Vet. Rec. 139, 443–445. Simpson, V.R., 2002. Wild animals as reservoirs of infectious diseases in the UK. Vet. J. 163, 128–146. Simpson, V.R., 2003. Sarcoptic mange in foxes. Vet. Rec. 152, 215– 216. Simpson, V.R., Neal, C., 1982. Angiostrongylus vasorum infection in snails and slugs. Vet. Rec. 111, 303–304. Skirnisson, K., Eydal, M., Gunnarsson, E., Hersteinsson, P., 1993. Parasites of the arctic fox (Alopex lagopus) in Iceland. J. Wildl. Dis. 29, 440–446. Sukhdeo, M.V.K., Bansemir, A.D., 1996. Critical resources that influence habitat selection decisions by gastrointestinal helminth parasites. Int. J. Parasitol. 26, 483–498. Taylor, M.A., Coop, R.L., Wall, R.L., 2007. Veterinary Parasitology, 3rd edition. Blackwell, Oxford, pp. 395–397 and 410. Trees, A.J., 1987. Angiostrongylus vasorum in dogs in Wales. Vet. Rec. 120, 424–1424. Webbon, C.C., Baker, P.J., Harris, S., 2004. Faecal density counts for monitoring changes in red fox numbers in rural Britain. J. Appl. Ecol. 41, 768–779. Wilkinson, D., Smith, G.C., 2001. A preliminary survey for changes in urban fox (Vulpes vulpes) densities in England and Wales, and implications for rabies control. Mammal Rev. 31, 107–110. Willingham, A.L., Ockens, N.W., Kapel, C.M.O., Monrad, J., 1996. A helminthological survey of wild red foxes (Vulpes vulpes) from the metropolitan area of Copenhagen. J. Helminthol. 70, 259–263. Wilson, K., Bjørnstad, O.N., Dobson, A.P., Merler, S., Poglayen, G., Randolph, S.E., Read, A.F., Skorping, A., 2002. Heterogeneities in macroparasite infections: patterns and processes. In: Hudson, P.J., Rizzoli, A., Grenfell, B.T., Heesterbeek, H., Dobson, A.P. (Eds.), The Ecology of Wildlife Diseases. Oxford University Press.
Veterinary Parasitology 154 (2008) 48–57 www.elsevier.com/locate/vetpar
Angiostrongylus vasorum and Eucoleus aerophilus in foxes (Vulpes vulpes) in Great Britain E.R. Morgan a,*, A. Tomlinson b, S. Hunter b, T. Nichols a,c, E. Roberts c, M.T. Fox c, M.A. Taylor b,c a
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK b Central Science Laboratory, Sand Hutton, York, UK c Royal Veterinary College, London, UK
Received 20 November 2007; received in revised form 19 February 2008; accepted 25 February 2008
Abstract The nematode parasite Angiostrongylus vasorum is a source of increasing concern in several parts of the world, where it causes significant disease in dogs. Wild canids, especially foxes, are likely to have a role in the epidemiology of canine infection, and the parasite could also affect fox health and population dynamics. The heart and pulmonary vasculature of 546 foxes culled mostly by gamekeepers in Great Britain in 2005–2006 were examined by dissection and a modified flushing technique. Forty foxes were found to be infected, giving an overall prevalence in the UK fox population of 7.3% (5.3–9.9). Prevalence varied widely between regions, from 0% (0–3) in Scotland and northern England to 23% (16–32) in south-east England. This closely matches the perceived incidence of disease in dogs, which is commonly diagnosed in the south-east but rarely in the north. In the Midlands, where disease has recently appeared in dogs, prevalence in foxes was 4.8% (2–11). Close geographical overlap of parasite distribution in foxes and dogs does not necessarily indicate an important wildlife reservoir of infection, but does suggest that A. vasorum might be spreading northwards. The hearts of infected foxes had thicker right ventricles than those of uninfected foxes, suggesting that the parasite could affect fox health and fitness. Burdens ranged from 1 to 59 adult nematodes. Sex, age and body condition were not significantly associated with infection. Eucoleus aerophilus and Crenosoma vulpis, nematode parasites of the respiratory system, were found in 213 and 11 foxes respectively, with slightly higher prevalence of E. aerophilus in the south and east. No specimens of the heartworm Dirofilaria immitis were found, giving an upper 95% confidence interval for prevalence of 0.84%. # 2008 Elsevier B.V. All rights reserved. Keywords: Parasite; Nematode; Cardiopulmonary; Lungworm; Capillaria; Crenosoma; Angiostrongylosis; Wildlife disease; Reservoir; Epidemiology
1. Introduction The nematode Angiostrongylus vasorum (Baillet, 1866) is parasitic in the right ventricle and pulmonary arteries of the red fox (Vulpes vulpes) and dog (Canis familiaris) (Bolt et al., 1994), and also infects the wolf
* Corresponding author. Tel.: +44 117 9287485. E-mail address: [email protected] (E.R. Morgan). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.02.030
(Canis lupus) (Segovia et al., 2001), coyote (Canis latrans) (Bourque et al., 2005) and other canid, mustelid and even felid species (Jeffery et al., 2004). Its distribution is worldwide but patchy, with ‘hyperendemic’ areas of high prevalence surrounded by regions in which the parasite is rarely found. In recent years, A. vasorum has increasingly been recorded outside these known hyper-endemic foci (Morgan et al., 2005). This is of great concern to veterinarians and dog owners, since infection with A. vasorum can cause
E.R. Morgan et al. / Veterinary Parasitology 154 (2008) 48–57
severe disease in dogs (Chapman et al., 2004; Koch and Willesen, in press). It is widely assumed that fox populations act as a reservoir of infection for dogs (Bolt et al., 1992), although the relative contribution of larvae shed by dogs and by foxes to canine infection has yet to be determined. Pathological changes have been observed in infected foxes (Poli et al., 1984, 1991; Simpson, 1996), but the effect of A. vasorum on vulpine health and population dynamics similarly remains unknown. In the UK, autocthonous infection of dogs with A. vasorum was first recorded in Cornwall (Simpson and Neal, 1982), with further cases appearing in south Wales (Patteson et al., 1987; Trees, 1987). The parasite was considered to be largely confined to these areas (Patteson et al., 1993; Martin et al., 1993) until increasing numbers of cases in south-east England confirmed significant spread to this area (Kriek, 2001; Chapman et al., 2004). Recent anecdotal reports of canine disease suggest that A. vasorum is expanding its range northwards. However, such reports are unreliable indicators of parasite distribution since parasitological diagnosis is rarely confirmed, and movement of dogs often makes it impossible to define exactly where they were infected. Foxes infected with A. vasorum were found close to the focus of canine disease in Cornwall (Simpson, 1996), but systematic data on distribution and levels of infection in foxes in the UK are absent. The present study aimed to determine the extent to which A. vasorum has expanded its range outside existing hyper-endemic areas of Great Britain, so that the risks of canine infection can be geographically defined. Associations between infection and various host factors were also examined in order to advance understanding of parasite epidemiology. Similar questions were addressed in relation to the nematode Eucoleus aerophilus (syn. Capillaria aerophila), which is parasitic in the respiratory tract and was coincidentally recovered, along with Crenosoma vulpis, when dissecting lungs for A. vasorum. 2. Materials and methods 2.1. Study animals Hearts and lungs were obtained from a total of 546 foxes (V. vulpes) culled as part of pest control programmes in different parts of Great Britain from January 2005 to April 2006. Sampling was opportunistic and some regions, including south Wales and Cornwall, and urban foxes, were under-represented. The foxes were shot and the carcases collected within
49
1–2 days. Foxes were classified by sex, age (young if incisors clean and unworn; adult if incisors showed loss of lobation and brown dentin spots on occlusal surfaces) and body condition (good, fair or poor, on the basis of lumbar and retroperitoneal fat thickness). The heart and lungs were removed together, taking care not to rupture the pulmonary trunk, and frozen at 20 8C. 2.2. Parasite recovery (heart) Hearts and lungs were dissected over the next 6–15 months, and extraction of A. vasorum was prioritised over that of other species. After exteriorisation from the pericardium, both ventricles were incised transversely mid way between the base and the apex of the heart. In a sub-set of hearts, the maximum thickness of the outer wall of each ventricle at this point was measured using callipers. The heart was separated from the lungs by transecting the major vessels as close as possible to the lungs, and all chambers as well as the pulmonary arterial trunk opened and inspected visually for nematodes. Blood clots were broken up by digital pressure and the heart chambers rinsed out over a tray, as well as the plastic bag in which the organs had been contained. The washings were inspected visually in the tray before being passed through sieves of 150 and 38 mm aperture. The residue on each sieve was resuspended in water and examined for nematodes under a dissecting microscope. The trachea was opened longitudinally and inspected closely for nematodes, which were removed, and a scalpel scraping of the mucosal surface was smeared onto a slide and examined under a transmission microscope at total magnification 100 for nematode eggs and larvae. 2.3. Parasite recovery (lungs) The lung lobes were separated into left and right groups. One side was selected at random for further dissection, and the other for flushing. Dissection consisted of opening all visible pulmonary vessels down to the narrowest practicable point (about 0.5 mm diameter) and picking out any nematodes seen. Thereafter, bronchi were likewise dissected and then rinsed with water over a sieve of 38 mm aperture, with the washings examined under the dissecting microscope. Lobes designated for flushing were separated into cranial and caudal groups and a modification of Jeffery’s (2004) technique applied. The largest blood vessel in each was identified and a pipette tip inserted firmly into the lumen and attached to a rubber hose. Water was pumped through using tap pressure and
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E.R. Morgan et al. / Veterinary Parasitology 154 (2008) 48–57
collected as it oozed out of the lungs and airways. Flushing continued until the lungs appeared pale and devoid of blood and the fluid appeared clear. If any parts of the lung appeared under-perfused the pipette tip was moved to a new vessel and flushing continued. The washings were sieved and examined as before. After flushing, the blood vessels and airways were dissected to their smallest practicable diameter and any nematodes picked out. No difference was found in the rate of recovery of Angiostrongylus using dissection or flushing followed by dissection after the first 200 foxes, but Eucoleus and Crenosoma were more often recovered from the flushed lobes. Subsequent lungs were therefore processed by flushing followed by dissection of all lobes. Nematodes were mounted on a microscope slide in water and identified under the transmission microscope, before being frozen in normal saline. The three nematode species known to occur in the cardiopulmonary tract of foxes in the UK – A. vasorum, C. vulpis and E. aerophilus (syn. C. aerophila) – are easily distinguishable on basic morphology (Taylor et al., 2007). 2.4. Statistical analyses Associations between the presence of nematodes and fox sex, age, body condition, geographical location and season were assessed separately for A. vasorum and E. aerophilus using logistic regression. Association between burdens of each species was estimated by Spearman rank correlation. Regions were defined by dividing Ordnance Survey National Grid references into arbitrary categories representing climatically different parts of the country. The prevalence of parasitism was compared across regions and seasons using the Chisquare test for association, and parasite abundance and intensity of infection using Kruskal–Wallis tests on raw data and one-way ANOVA on log10 (x + 1) transformed data (Ro´zsa et al., 2000). Observer bias was examined by Chi-square comparison of observed and expected frequency of parasite occurrence. 95% Confidence
intervals for prevalence were estimated using the exact binomial method. The geographical distribution of A. vasorum was assessed for clustering using Kulldorff’s spatial scan statistic, within a purely spatial Bernoulli model. Spatial autocorrelation was assessed by Moran’s I statistic, with adjacency defined as shared boundaries at the county level. Statistical analyses were conducted using SPSS 12.0 (SPSS Inc., Chicago), ArcGIS 9.x (ESRI, California), SatScan v7.0.3 (SaTScanTM, Boston) and GeoDa version 0.9.5i (Spatial Analysis Lab, Illinois) computer packages. 3. Results 3.1. Parasite identification and overall prevalence Nematodes were found in 44% (95% confidence intervals, CI 39.7–48.1) of foxes. E. aerophilus was by far the most common species, followed by A. vasorum (Table 1). Too few C. vulpis were found to justify inclusion of this species in further analyses. Body condition was also excluded given that there were no animals in poor condition and only 23 in fair condition, and preliminary analysis showed that condition was not significantly associated with prevalence of any nematode species. Logistic regression revealed that A. vasorum was highly significantly over-represented in the south-east (Table 2). A. vasorum was equally common in both sexes, but E. aerophilus was more common in males than in females (prevalence = 0.61, CI 0.52–0.69 in males and 0.41, CI 0.32–0.50 in females). E. aerophilus prevalence also varied by region, being more common in the southern and eastern regions. Infection was not significantly associated with age category for either nematode species. 3.2. Anatomical location and extraction Only 41% of the A. vasorum recovered were found in the heart and pulmonary trunk artery, the rest being recovered from the smaller vessels by flushing and fine
Table 1 Nematodes found in the cardiopulmonary tract of 546 foxes Species
Angiostrongylus vasorum Crenosoma vulpis Dirofilaria immitis Eucoleus aerophilus
Prevalence
0.07 0.02 0 0.39
CI = exact binomial 95% confidence interval.
CI
0.05–0.10 0.01–0.03 0.00–0.01 0.35–0.44
Intensity Mean
Variance
Range
6.7 5.6 – 4.4
88.1 54.0 – 40.5
1–59 1–22 – 1–51
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51
Table 2 Composition of the binary logistic regression model and significant predictors for nematode infection Variable
d.f.
Category
n
(a) Model constituents Region
4
North Midlands South East South-East
138 126 29 128 125
Season
3
Winter (December to February) Spring (March to May) Summer (June to August) Autumn (September to November)
120 190 90 146
Sex
2
Not known Male Female
275 143 128
Age
2
Not known Young (<1 year) Adult (>1 year)
230 265 51
Factor
d.f.
Wald statistic
p
Significant levels
(b) Significant predictors of A. vasorum infection. HR = 2.951, 8 d.f., p = 0.937 Region 4 27.120 <0.001 South-East
p
OR
CI
<0.001
6.75
2.51–18.2
Summer Autumn
0.019 0.029
5.00 3.45
1.31–19.2 1.14–10.5
(c) Significant predictors of E. aerophilus infection. HR = 0.624, 8 d.f., p = 0.624. Region 4 11.101 0.025 South East
0.031 0.002
2.63 2.40
1.09–6.33 1.38–4.18
0.001
2.44
1.47–4.06
Season
Sex
3
2
8.551
29.830
0.036
<0.001
Male
HR = Hosmer–Lemeshow goodness of fit Chi-square statistic, d.f. = degrees of freedom, OR = odds ratio and CI = 95% confidence intervals.
dissection. Of foxes ultimately determined to be positive for A. vasorum, only 36% would have been detected by dissection of the heart and pulmonary trunk alone. Larvae were found on the tracheal scrape in only 10% of infected animals. There was no correlation between parasite burden and the proportion of A. vasorum found in the smaller pulmonary vasculature, and no significant difference in the number recovered from the lungs by dissection and flushing. In several cases nematodes were dissected from the airways after flushing had been completed. Of animals positive for E. aerophilus, 55% were detected by gross dissection of the trachea and bronchi (of which 62% were also positive on flushing), and a further 34% by recovery of adults or fragments of adults in the lung flush, with the remainder detected only by observation of eggs on the tracheal scrape. However, eggs were detected in the trachea of only 26% of foxes that carried adult E. aerophilus, even though around half of the infrapopulations included gravid female worms. Just a single adult worm was found in 37% of animals positive for E. aerophilus, and two worms in 18%. For A.
vasorum, 18% of infrapopulations comprised a single adult or immature worm. 3.3. Geographical distribution The broad-scale geographical distribution of the two most common nematode species is summarised in Table 3. Although very few C. vulpis were recovered, this species was found in three of the five regions, and 6 of the 11 infected foxes were from the North. E. aerophilus was fairly evenly distributed between regions, but the occurrence of A. vasorum was more varied (Fig. 1). Most of the infected foxes were found in south-east England, and groups of infected foxes in the south, east and Midlands were interspersed by areas of low prevalence. No A. vasorum were recovered from foxes in the far north of England or in Scotland. Spatial analysis pointed to a significant cluster of infection in the south-east, centred on National Grid Reference SU 200500 and with a radius of 84.4 km. Within this area 38 foxes were sampled, of which 20 were infected compared with an expected
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E.R. Morgan et al. / Veterinary Parasitology 154 (2008) 48–57
Table 3 Regional variation in the prevalence of cardiopulmonary nematodes of foxes in Great Britain Region
North Midlands South East South-east
OS National grid squares
NJ-NY, SD-SJ SK, SO, SP ST, SY TA-TM SU, SZ, TQ-TV
Total
A. vasorum
E. aerophilus
Prevalence (%)
CI
Prevalence
CI
0 4.8 6.9 1.6 23.2
0–3.0 2.0–11 1.2–24 0.2–6.2 16–31
40 39 55 45 30
32–49 30–49 36–73 37–54 23–39
7.1
5.2–9.7
40
36–44
OS = ordnance survey. See Table 2 for sample sizes.
number of 3 (relative risk 14.044, log likelihood ratio 33.1, p < 0.001). Spatial autocorrelation in prevalence between counties was of borderline significance (Moran’s I = 0.0694, p = 0.086) (Fig. 2).
Fig. 1. The geographical distribution of Angiostrongylus vasorum in foxes in Great Britain. Clear circles represent foxes clear of infection, and filled squares infected individuals. Some markers overlie each other where foxes were collected from the same location. The figure illustrates the relative distribution of sampling effort and positive samples. See text for formal spatial analysis.
Fig. 2. Prevalence of A. vasorum by county. Counties not sampled are not distinguished from those with zero prevalence (see Fig. 1). The sampling design was non-probabilistic and prevalence is indicative only, see text for formal comparison of regional occurrence.
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Patterns in prevalence were generally echoed by parasite abundance and to a lesser extent by the intensity of infection. All species were overdispersed in distribution among hosts, with variance in abundance greatly exceeding the mean (Table 1). A. vasorum abundance differed significantly between regions (Kruskall–Wallis x2 = 66.7, 4 d.f., p < 0.001; ANOVA F4, 541 = 16.876, p < 0.001), and was higher in the south-east than in all other regions (Tukey’s pairwise comparisons p 0.004). Intensity of infection was also higher in the south-east (mean 7.6, variance 113.7) than in other regions (range 0– 5.0), but the difference was not significant. 3.4. Seasonal patterns A. vasorum was more commonly found in foxes killed in summer and autumn (Table 2), but there were no significant seasonal differences in abundance. E. aerophilus prevalence did not vary by season. 3.5. Parasite associations There was no association between infection with E. aerophilus and A. vasorum (x2 = 0.051, 1 d.f., p > 0.50), and no correlation between the burden of each species in individual foxes (Spearman r = 0.02, p = 0.58). 3.6. Cardiopulmonary pathology Although the mean thickness of the left ventricle was not significantly different between infected and noninfected groups (overall mean 17.48 mm S.E. 0.31, n = 34), the right ventricle was significantly thicker in infected foxes (infected 7.1 mm S.E. 0.22, n = 17; uninfected 5.0 0.16, n = 17; mean difference 2.1 0.27, t = 7.71, 32 d.f., p < 0.001). The ratio of right to left ventricular thickness was also greater in infected than in uninfected foxes (0.43 0.02 cf. 0.28 0.01, t = 8.97, 32 d.f., p < 0.001). Histological examination of lung tissue from foxes infected (n = 10) and uninfected (n = 10) with A. vasorum suggested that parasitism was associated with breakdown in alveolar structure (honeycomb lung) and inflammatory cell infiltration. However, lung structure was also disrupted in uninfected foxes, presumably as a result of the repeated freezing and thawing of hearts and associated autolysis, so observed changes are not described further. 3.7. Observer bias Eight identically trained observers dissected the hearts and lungs, each contributing between 22 and 95
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samples to the total. The frequency with which each observer found nematodes did not differ significantly from that expected by chance for A. vasorum (x2 = 7.119, 7 d.f., p > 0.10), but there was significant observer bias for E. aerophilus (x2 = 42.84, 7 d.f., p < 0.001). Within-observer prevalence for this species varied from 13 to 61%, and two observers detected E. aerophilus more often than expected. Since samples were drawn fairly randomly from the stored material, this did not lead to bias in regional prevalence or other risk factors for infection, so data were analysed in full without adjustment. A sub-sample of 137 negative hearts and lungs were frozen after dissection and thoroughly re-examined by different observers several months later. Four of these samples contained 1 or 2 adult A. vasorum that had been previously undetected. 4. Discussion Since two of the three known endemic areas for A. vasorum in the UK (Cornwall and south-west Wales) were not sampled, prior expectation in the present study was to find this species only in south-east England. In fact, low levels of infection were also discovered in foxes in other parts of southern and eastern England and the Midlands. The lack of previous studies on this scale makes it impossible to demonstrate that the parasite is spreading northwards, although our data and the expanding range from which canine cases are reported are consistent with this hypothesis. Whether foxes act as the main reservoir of infection for dogs or not, veterinarians should be aware of the possibility of canine angiostrongylosis outside areas in which it has previously been diagnosed, since the parasite is present and transmission is clearly possible across a large part of the UK. Although the foxes sampled here were mostly rural and culled by gamekeepers, urban and sub-urban foxes presumably have greater opportunities for contact with dogs and could be more important in terms of disease transmission across the wildlife-domestic animal boundary. This appears to be the case for the better studied cestode parasite Echinococcus multilocularis (Deplazes et al., 2004; Morgan et al., 2004). In Denmark, the highest prevalence of A. vasorum was found in urban and suburban foxes in Copenhagen (Saeed et al., 2006). Future studies should aim to sample urban as well as rural foxes for parasites, for example, through examination of road-killed animals or fresh faeces. A. vasorum was not found in foxes north of the Midlands, and although the possibility of foci of infection above this latitude cannot be excluded, prevalence is clearly much lower in the north than
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the south of Great Britain. However, it is not clear whether northern areas are relatively unsuitable for transmission, or whether they could sustain a parasite population but have yet to be colonised. Given the frequent long-range movement of dogs within the UK, if conditions exist to support the parasite it can only be a matter of time before it becomes established in all suitable regions. The introduction of infection by dogs and establishment in local fox populations is considered to underlie the spread of A. vasorum in Scandinavia ˚ blad et al., 2003; Saeed et al., 2006). Unfortunately, (A too little is known about the parasite’s climatic and biological requirements to make sensible predictions about areas at risk from range expansion. The intermediate host range is broad (Guilhon and Cens, 1973) and northern parts of Great Britain certainly harbour competent slug and snail vectors. C. vulpis, which also uses gastropod mollusc intermediate hosts, was found in several foxes in the Scottish borders. On the Canadian island of Newfoundland, C. vulpis is widely distributed in foxes, but A. vasorum inhabits only areas with a mean winter temperature above 4 8C (Jeffery et al., 2004). Since this applies to the whole of Great Britain, it seems likely that there is no absolute climatic obstacle to the establishment of either species, although higher gastropod densities in mild and humid parts of the country may favour transmission (Simpson and Neal, 1982). A. vasorum has been found in foxes in a wide range of countries with differing climates (Bolt et al., 1994), and the reasons for its apparently patchy distribution in the UK and elsewhere are unknown. In this context, the marginal level of autocorrelation in A. vasorum prevalence found at county level in this study, along with the single identifiable cluster of infection in the south-east, is interesting. If spread were by means of organic expansion of established hyper-endemic foci, stronger autocorrelation would be expected, at least until foci begin to coalesce. Our results might indicate that the focal geographical distribution of A. vasorum in the UK has already broken down into a more generalised colonisation of the southern half of the country. However, the arbitrary nature of the county boundaries, the limited sample size, and especially the omission of foxes from south-west Wales and Cornwall, limit the strength of the conclusions that can be drawn. Ultimately, tracking parasite spread will require longitudinal studies. In Denmark, the abundance of E. aerophilus, but not A. vasorum, was found to correlate with fox population density (Saeed et al., 2006). The lack of detailed estimates of fox density in the UK and most other countries (Webbon et al., 2004) limits the predictive
value of this parameter, while threshold host densities for A. vasorum persistence are not known. The emergence of A. vasorum in southern England in the 1990s coincided with an outbreak of sarcoptic mange that reduced fox populations locally by as much as 90% (Baker et al., 2000; Simpson, 2002), although in some areas urban fox populations appeared to increase during this period (Wilkinson and Smith, 2001). The importance of fox population density in enabling the parasite to become established is therefore unclear, while other effects such as increased dispersal (Baker et al., 2000) and possibly immuno-suppression could exacerbate parasite spread during mange outbreaks even as overall population density decreases. It is perhaps worth noting that several European countries have experienced outbreaks of sarcoptic mange in foxes in recent years (Simpson, 2003), including Spain and Denmark, which also have locally high prevalence of A. vasorum (Willingham et al., 1996; Segovia et al., 2004). Sarcoptic mange has been found to be associated with the presence of helminths in foxes in Italy (Balestrieri et al., 2006). It is likely that interactions between climate and intermediate and definitive host densities influence the chances of parasite establishment and persistence in new areas. In this context, climate change and associated alterations in slug and snail phenology (Root et al., 2003) could affect the global distribution of A. vasorum and the local risk of infection for foxes and dogs. E. aerophilus is clearly common in foxes in all parts of Great Britain. Regional differences appear to exist, but are smaller in magnitude than observer variation and must be considered slight at most. This apparent stability in level of infection is also a feature elsewhere in Europe, where prevalence in foxes is typically between 35 and 65% (Manˇas et al., 2005). The parasite appears to be more common in Denmark (74% prevalence; Saeed et al., 2006) and Norway (88%; Davidson et al., 2006), although recovery methods were different. In the present study, no relationship was found between season and risk of infection. This could be explained by lack of seasonality in transmission, parasite longevity that exceeds the timescale of seasonal effects, or damping of seasonal effects, for example by density dependence or immunity. E. aerophilus was found more often in male than in female foxes, in line with theories of immune handicap in male mammals (Wilson et al., 2002). Although no relationship was found between E. aerophilus and A. vasorum infection, other workers have recorded a positive correlation in burdens of these species, and a negative correlation between A. vasorum and C. vulpis, which they took to
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indicate that different intermediate host species are used (Saeed et al., 2006). Jeffery et al. (2004), in contrast, found no relationship between burdens of A. vasorum and C. vulpis in individual foxes. C. vulpis was rarely found in the present study, but this is not considered an accurate estimate of prevalence since the small size of the nematode and observer prioritisation of the larger species are likely to have decreased detection rate. Sensitivity of observed prevalence to method of dissection and observer experience might explain some of the wide variation in prevalence (5–75%) previously reported for C. vulpis in foxes in other parts of Europe (Davidson et al., 2006). The impact of cardiopulmonary nematode infection on fox health and population dynamics is unknown. C. vulpis and A. vasorum are well recognised causes of disease in domestic dogs (Martin et al., 1993; Shaw et al., 1996; Chapman et al., 2004), and have been associated with pulmonary pathology in foxes (Poli et al., 1991; Conboy and Adams, 1995). E. aerophilus has been found in a wide range of carnivore species including humans, sometimes in association with pathological changes in the lungs and airways (e.g. Crum et al., 1978; Morrison and Gier, 1978; Greenlee and Noone, 1984; Laux, 1987; Richardson et al., 1992; Skirnisson et al., 1993; Martin, 1998; Nevarez et al., 2005; Nelson et al., 2007; Krone et al., 2008; Lalosevic et al., 2008). The poor condition of the tissue in this study made detailed histopathology of dubious worth, so changes are not described in detail. However, the right ventricle of the heart was significantly thickened in animals in which A. vasorum was found. Poli et al. (1984) also noted right ventricular hypertrophy in infected foxes, although Jeffery et al. (2004) failed to find this even at much higher burdens than we observed, and in fact infection was associated with reduced overall heart weight relative to body weight. Jeffery et al. (2004), like us, found no difference in body condition between infected and uninfected foxes. The implications of A. vasorum infection for vulpine health and fitness are uncertain and would be difficult to measure. Cross-sectional studies are a notoriously insensitive way of detecting effects of parasitism on wildlife populations (Irvine, 2006), but, in the absence of sustained funding for wildlife disease surveillance and interventional studies, often constitute the only available data. The present study found an increased prevalence of angiostrongylosis in summer and autumn. A lack of any seasonal effect in most other published surveys can be explained by concentrated collection of carcases during short hunting seasons, and consequently limited
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seasonal range. Given the long pre-patent period of A. vasorum, the observed pattern could reflect increased transmission in spring and summer. Foxes are likely to include more amphibians in their diets at this time of year, providing greater opportunity for infection by ingestion of paratenic hosts (Bolt et al., 1993, 1994). Season is confounded by age in foxes, since cubs are born in spring, but the observed seasonal pattern cannot be explained by age-dependent infection since there was no difference in A. vasorum prevalence or intensity between age categories. It is perhaps surprising that no age effect was found for either A. vasorum or E. aerophilus. This could be explained by different combinations of age-dependent acquisition, acquired immunity and parasite-induced host mortality. However, cross-sectional data, especially at such crude age resolution, constitute a blunt tool for the detection of such dynamics. It should also be borne in mind that the prevalence and intensity of infection with A. vasorum were lower in this study than in comparable surveys in Denmark (49%, mean intensity 7.4; Saeed et al., 2006) and Newfoundland, Canada (56%, 230; Jeffery et al., 2004), and host-parasite relationships could be radically different at higher levels of infection. Indeed, Jeffery et al. (2004) considered the lack of a positive age– intensity relationship to indicate that immunity was limiting burdens of A. vasorum in foxes in Newfoundland, while C. vulpis intensity actually decreased with age. At lower overall parasite abundance in Denmark, prevalence of A. vasorum (and also E. aerophilus) increased with age, although mean intensity did not change (Saeed et al., 2006). Higher intensity of infection could also affect anatomical location of the adult parasites: thus, Jeffery found that 78% of infected foxes had A. vasorum in the heart, compared with 36% in the present study, which might be explained by competitive displacement of worms from the arteries at high infrapopulation size (Sukhdeo and Bansemir, 1996). Flushing the lungs and then dissecting the pulmonary arteries was found to be an effective way of detecting A. vasorum, although it is difficult to concurrently maximise the detection rates of all species of cardiopulmonary nematode. Importantly, surveys of A. vasorum that are limited to dissection of the heart and major pulmonary vessels are likely to seriously underestimate both the prevalence and abundance of this species. The flushing technique causes physical disruption of the lung tissue and is less suitable for studies in which the anatomical position of the nematodes and the structure of the tissue are important. It is also time-consuming, taking experienced investigators around 1 h per animal. Given
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that coarse dissection of the heart and pulmonary trunk is some 10 times quicker, the reduced sensitivity of this method might be offset by increased sample size when investigator time rather than access to fox carcases is the major limiting factor, and the important variable is presence of the parasite rather than accurate estimates of prevalence or abundance. It is also notable that in the present study the main patterns in relation to geographical distribution, season and host sex were evident using prevalence data alone. Analysis of abundance and intensity generally reinforced these patterns but was less sensitive in detecting them. From our data, prevalence therefore appears to be the most useful single variable in detecting patterns in level of parasitism within populations, even though it is very sensitive to differences in detection efficiency between studies. The present study demonstrates that A. vasorum is more widespread in foxes in Great Britain than previously supposed, and suggests that northward spread is likely. Our data provide an important baseline for future studies. As well as surveying parasite distribution, future work should attempt to elucidate the factors important to parasite transmission and abundance so that the risks of range expansion can be more precisely assessed. Acknowledgements We are grateful for the assistance of Colin Morgan, Vicky Jackson and other CSL staff in organising the fox collection and sample storage, and of Nicholas Ilchyshyn, Tanya Kahrs, Peter Ward, Madeleine Krajewski, Hannah Ridout, William Low, Charlotte Young, Dhanurjaya Thyagaraja, Gemma Jones and especially Hannah Rose and Kira Sawyer in conducting the dissections. The Nuffield Trust and the Wellcome Trust provided financial support for summer studentships. Fox collections were facilitated as part of an ongoing project on trichinellosis funded by the UK Food Standards Agency (FSA). We thank Vic Simpson for his encouragement and useful discussions throughout this work. References ˚ blad, B., Christensson, D., Lind, E.O., Agren, E., Morner, T., 2003. A Angiostrongylus vasorum etablerad i Sverige. Sven. Veterina¨rtidning 12, 11–15 (In Swedish). Baker, P.J., Funk, S.M., Harris, S., White, P.C.L., 2000. Flexible spatial organization of urban foxes, Vulpes vulpes, before and during an outbreak of sarcoptic mange. Anim. Behav. 59, 127– 146.
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