Importance of concentration tests in diagnosing heartworm in dogs

Author : Walter Tarello, veterinary surgeon,
C.P. 42, 06061 Castiglione del Lago
PERUGIA (Italy)
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ABSTRACT

The aim of this study was to compare the concentration and serological tests results in two groups of 7 dogs each, with heartworm infection, and living in endemic and non-endemic areas. All the 14 dogs had microfilariae of Dirofilaria immitis in the blood, detected by a filtration test (Difil-test). Differently, serological results were always negative in dogs living in non-endemic area and in 2 out of 7 dogs from endemic-area. These results suggest that false seronegativity seems easy to occur in no or low-endemic areas. Consequently, the search of microfilariae by Knott or filtration tests is still strongly recommended in such places , preferably combined with serological tests.

INTRODUCTION

At the present time, heartworm disease caused by the filarial helminth Dirofilaria immitis is not endemic in most of the European countries (Greeve et al., 1983; Schrey, 1996). All cases reported in UK, the Netherlands, Sweden, Switzerland, Austria, and Germany, have been imported previously from endemic areas (Schrey, 1996; Zahler et al., 1997; Bucklar et al., 1998). In Germany, canine dirofilariasis has been discovered on routine blood tests for microfilaria in 13% of dogs with history of travel to Italy, Portugal, Spain and Corsica and in 10% of dogs imported from Italy, Spain and Portugal (Schrey, 1996).

During the last 20 years the infestation has spread to colder areas where suitable vectors exist, such as Bretagne in France (Doby et al., 1986), Switzerland (Bucklar et al., 1998) and northern provinces in Italy (Genchi et al. 1995; Rossi et al., 1996). The absence of large number of suitable vectors in the UK probably explains why the disease has not yet become endemic in the country, despite importation of infected dogs (Murdock, 1984).

In Italy, endemic areas are the Po plane and the Tuscan coast (Fig. 5), meanwhile the remaining parts of the country are considered free or low endemic areas (Genchi et al., 1995; Rossi et al., 1996). Introduction of the disease to such areas is thought to have resulted initially from the free movement of infected animals from warmer, higher incidence areas. Diagnosis of canine dirofilariosis in animals which originate from a low-endemic region is difficult for 3 main reasons:

1) possible absence of circulating microfilariae (occult disease),
2) high frequency of a low number of adults, which in turn produces false negative results, using either ELISA or latex agglutination test,
3) possible false sero-positive results, due to cross-reaction with adult antigens of Dirofilaria (Nochtiella) repens (Schrey, 1996), the agent of subcutaneous dirofilariasis causing chronic pruritic dermatitis in dogs (Tarello , 1999).

It is generally thought that concentration tests (Knott, filtration) are the most sensitive, because detection of microfilaria in the blood is diagnostic for the presence of adults in the pulmonary arteries (BSAVA, 1998). This observation is particularly true for low-endemic or newly colonized areas , where a low number of adults (false seronegatives) and/or a hight prevalence of Dirofilaria repens (false seropositives) are to be observed (Genchi et al., 1995).

A negative concentration test, does not however, rule out a diagnosis of dirofilariosis because reports say that 20 to 30 per cent of canine infections and the majority of infected cats are negative for microfilaria (Davoust and Ducos de Lahitte, 1989; Rawlings and McCall, 1982; Rawlings and Calvert, 1995). Consequently, occult disease can be diagnosed by tests using monoclonal antibodies to circulating adult antigens (ELISA or latex agglutination).

The first aim of this study was comparing the concentration and serological tests results, obtained from 2 samples of dogs with heartworm patent infection, in an endemic (Alexandria, Piedmont) and a non-endemic area (Fermo, Marche) of Italy (Fig. 5).

The second aim was that of determining how correlated are the two different test techniques in not endemic areas, where suspected cases are not often encountered and familiar to the clinicians, their diagnosis is much more difficult on objective grounds, and missing cases can contribute to spreading the infestation among dogs, in association with lack of preventive medication.

MATERIAL AND METHODS

Fourteen adult dogs from two different areas and with a diagnosis of heartworm disease, were examined on the basis of laboratory tests results for Dirofilaria immitis infection. Seven symptomatic canine dirofilariasis were collected in 1998, during a 8-months period (spring to autumn) of clinical practice in highly endemic area (Alessandria, Piedmont; Fig. 5). The apparently low number of registered cases is justifyied by the widespread use of prophylaxis (Ivermectine, Milbemycin oxime) in that region. The other seven patent cases of heartworm disease were collected in 1999 during a 8-months period (spring to autumn) of practice in a non-endemic area (Fermo, Ascoli Piceno province) of Italy, where prophylaxis is fairly rare (Fig. 5).

No selection was made in admitting recorded cases to the present study, but the physical examination had to reveal at least one
symptom of dirofilariasis : cough, weight loss, weakness (BSAVA, 1998). Two specimens (2+2 ml.), an whole blood sample in EDTA and a serum sample, were collected from each dog, and submitted to concentration test (Difil-test) and to canine heartworm antigen test (Wittness Dirofilaria, Merial). The samples for the differential identification of microfilariae were fixed using 2 per cent formalin, filtered and examined by light microscopy at x10 and x40 magnifications. The 7 samples from the non-endemic area (Fermo) were subsequently tested for the heartworm antigens using PetCheck (IDEXX Laboratories) and Wittness Dirofilaria (Merial).
No routine clinical haematology tests or radiographic abnormalities are diagnostics for dirofilariasis (Rawlings and Calvert, 1995). Thereby these examinations were not considered. Medical treatment followed any diagnosis of heartworm disease.

RESULTS

Microfilariae of D. immitis (1 to 9 per sample) were detected in all the seven dogs with patent heartworm disease living in the highly endemic area of Alessandria (Piedmont). Serological results were positive in 5 out of 7 animals.
In the non-endemic area of Fermo (Ascoli Piceno province), concentration test results were similar and microfilaraemia due to D. immitis (1 to 8 per sample) was present in the total number of dogs with clinical manifestations (Fig.1,2,4), without significant quantitative differences in comparison with the results coming from the endemic area. Unexpectedly, in contrast with these microscopical findings, serological results in this group of dogs were always negative , using subsequently both PetChek and Wittness Dirofilaria tests for canine heartworm antigen detection .

It is interesting to note that animals so tested were all symptomatic: coughing was the principal sign (13/14), followed by poor appetite (5/14), vomiting (3/14), weight loss (3/14), weakness (2/14) and collapse (1/14).

No particular differences in severity and duration of symptoms could be detected between the two canine groups. Complete resolution followed medical therapy (melarsomine dihydrochloride, aspirin, ivermectin) in 6 out of 7 dogs from the endemic area. One 13-years-old mixed male, with high number of microfilariae and antigens-positive, died during the therapy.

Comparatively, 5 dogs from the low endemic area experienced a complete remission after identical treatment, meanwhile 2 dogs died.

DISCUSSION

Comparison between direct identification of microfilariae and canine heartworm antigen tests in two groups of 7 + 7 symptomatics dogs has confirmed that concentrations tests (Difil-test) are the most sensitive and, furthermore, that they appear particularly useful in non-
endemic or newly colonized areas. As a matter of fact, none of seven dogs from a non-endemic area in Central Italy (Fermo, Marche), with D. immitis microfilariae (fig. 1,2,4) in the blood and patent disease, proved seropositive with two antigen tests (PetChek and Wittness). Differently, in the endemic area of North Italy (Alexandria, Piedmont), 5 out of 7 microfilaraemic and symptomatics dogs resulted seropositive (Witness).
Although the number of cases here reported is limited and statistical analysis is not possible, the results of the present study show that difficulties in heartworm diagnosis in non-endemic area may be due not only to the unfamiliarity of clinicians with the disease but, also, to false sero-negative diagnostic test results, apparently due to low number of adults nematodes carried by every infected dog.

The frequency with which this occurs, as a consequence also of increasing pet's travel, deserve further large-scale investigations.
In a recent study from another non-endemic area (Munchen, Germany), among 72 imported or travelling dogs with D. immitis microfilariae in the blood, and examined between 1993-96, 27 (37.5%) resulted sero-negative with the ELISA test (Zahler et al., 1997), apparently confirming the results here reported.

Two are the others canine filariases releasing microfilariae in the blood and described in Italy: Dirofilaria (Nochtiella) repens and Acanthocheilonema (syn.: Dipetalonema) reconditum (Pampiglione et al., 1995; Tarello, 1999).

Differential identification is made by light microscopy (x10,x40) using the concentration tests (Knott's test, Difil-test). When fixed using 2 per cent formalin, microfilariae of D. immitis are 262-338 mm in length and 4-6,2 mm in width (Schrey, 1996), distinguishing them from the non-pathogenic filarial larvae of A. reconditum which are shorter and thinner ( 241-287 mm x 3,8-5 mm). Furthermore, the tails of A. reconditum microfilariae are frequently curved as a ovariectomy hook (Fig. 3). Larvae of Dirofilaria (Nochtiella) repens are the greatest, 270-379 mm in lenth and 6-8,3 mm in width (Pampiglione et al.,1995), with a characteristic cephalic end roundish, short and clear (Fig.6). Comparatively, the cephalic end of D. immitis microfilariae is cone-shaped and dark (Fig.1,2) and their tail is very thin (Fig.4).

Detection of D. immitis microfilariae in a seronegative dog, can occur when only a single gravid female lives in the pulmonary arteries of the host . In fact, according to diagnostics manufacturers, infestations due to males, non-gravid females or less than two gravid females are difficult to identify by serological tests and can produce false negativities (Schrey, 1996).

To which extent this happen in practice has never been properly investigated. The results of this preliminary study on a small number of animals naturally infected , indicate that false seronegativity seems easy to occur in non-endemic areas, which represents the major parts of italian and european territories. This observation suggests that the search for microfilariae must not be abandoned, and preferably coupled with serological tests, which are important tools in enzootic regions, providing semiquantitative data upon the parasitic burden of infection (Rawlings and Calvert, 1995). Combining the Knott test and an antigen test improves overall accuracy. For instances, Hoover et. al. (1996) obtained an accuracy of 95% when both the Knott test and the DiroCheck antigen test were used.

Geographic distribution of D. immitis and D. repens coincides (Fig. 5) in many areas of South Europe (Genchi et al., 1995; Rossi et al., 1996) and cross reactivity between the two nematodes is regularly shown by different tests, producing false seropositive results (Schrey, 1996). To prevent diagnostic errors, a concentration test must be performed even in such cases.

Occult disease, frequently reported at a rate of 20 to 30 per cent (Rawlings and McCall, 1982; Davoust and Ducos de Lahitte, 1989), was never observed during the 2-years period of the present work. Occult dirofilariosis is a particular aspect of large-scale epidemiological and routine screenings and its rate is largely dependant on non-objective factors, such as individual skills and experience in microfilariae detection. Furthermore, dogs included in this study for examinations were all symptomatics and this might well explain the percentage of microfilaraemia recorded and the absence of occult disease.

CONCLUSIONS

In summary, serological tests for heartworm disease are frequently negative in non-endemic areas because of the low number of D. immitis adults in the pulmonary arteries of the host. The presence of a single female and/or one to many males is hardly detected by these tests. Apparently, this condition is common in non-enzootic areas.

In such cases, accurate concentrations tests, however, can demonstrate circulating microfilariae in many dogs, avoiding wrong diagnosis and diffusion of dirofilariasis to newly colonized areas.

REFERENCES

BSAVA's Scientific Committee. (1998) Heartworm disease. Journal of Small Animal Practice. 39, 407-408.

Bucklar H, Scheu U, Mossi R, Deplazes P. (1998) Is dirofilariosis in dogs spreading in south Switzerland ? Schweiz Arch Tierheilkd. 140, 255-60

Davoust B, Ducos de Lahitte. (1989) Evolution de l'enzootie de dirofilariose dans les chenils militaires du sud est. Revue de Médecine Vétérinaire.140, 15-19.

Doby JM, Guiguen C, Lefevre R. (1986) Présence de Dirofilaria immitis (Leidy, 1856) chez le chien en Bretagne. Bulletin Société Francaise de Parasitologie. 4, 51-54.

Genchi C, Venco L, Magnino B, Di Sacco B, Perera L, Bandi C, Pignatelli P, Formaggini L, Mazzucchelli M. (1993) Aggiornamento epidemiologico sulla filariosi del cane e del gatto. Veterinaria. 2, suppl., 5-11.

Genchi C, Solari Bazano F, Bandi C, Di Sacco B, Venco L, Vezzoni A, Cancrini G. (1995) Factors influencing the spread of heartworm in Italy; interaction between Dirofilaria immitis and Dirofilaria repens . In: Proceedings of the Heartworm Symposium '95 . Soll M.D., Knight D. II, eds., American Heartworm Society, Batavia, IL, 65-71.

Greeve RB, Lok JB, Glickman LT. (1983) Epidemiology of canine heartworm infection. Epidemiology Revue. 5, 220-246.

Hoover JP, Campbell GA, Fox JC, Claypoo PL, Mullins SB. (1996) Comparison of eight diagnostic blood tests for heartworm infection in dogs. Canine Practice. 21, 11-24.

Murdoch DB. (1984) Heartworm in the United Kingdom. Journal of Small Animal Practice. 25, 299-305.

Pampiglione S, Canestri Trotti G, Rivasi F. (1995) Human dirofilariasis due to Dirofilaria (Nochtiella) repens : a review of world literature. Parassitologia. 37 :149-193.

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Rawlings CA, Calvert CA. (1995) Heartworm disease. In: E.S.J. Ettinger ESJ and Feldman EC. (4th ed.): Textbook of Veterinary Internal Medicine, W.B. Saunders, Philadelphia, 1046-1068.
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Schrey CF. (1996) Epidemiologische Fallanalyse der kardiovaskularen Dirofilariose (Herzwurmerkrankung) bei Hunden in Deutschland. Dissertation for the degree of Doctor of Veterinary Medicine, der Freien Universitat Berlin.

Tarello W. (1999) La dirofilariose sous-cutanée à Dirofilaria (Nochtiella) repens chez le chien. Revue bibliographique et cas clinique. Revue de Médecine Vétérinaire. 150, 691-702.

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FIGURES

1) Dirofilaria immitis microfilaria (x10, filtration test) in the blood of a seronegative symptomatic dog from the non-endemic area of Fermo (Marche, Italy)

2) Dirofilaria immitis microfilaria (x4) in a fresh blood smear from a seronegative dog , living in non-endemic area (Fermo, Italy).

3) Detail of the caudal end of a Achantocheilonema reconditum microfilaria (x40, filtration test).

4) Detail of the caudal end of a Dirofilaria immitis microfilaria (x40, filtration test), from a seronegative and symptomatic dog living in non-endemic area (Fermo, Marche, Italy).

5) Map of Italy, showing the endemic areas for Dirofilaria immitis (black) and for Dirofilaria (Nochtiella) repens (dots) and the towns of Alessandria and Fermo (Ascoli Piceno province).

6) Dirofilaria (Nochtiella) repens microfilaria (x10), the causative agent of subcutaneous dirorilariasis, associated with pruritic dermatitis in dogs.

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IMPORTANCE IN THE DOG OF CONCENTRATION TESTS FOR THE DIAGNOSIS OF HEARTWORM DISEASE IN NON ENDEMIC AREAS.