Novel Molecular Assay for Simultaneous Identification of Neglected Lungworms and Heartworms Affecting Cats

Abstract

Feline lungworms and heartworms are stimulating the interest of the scientific community due to their clinical impact and apparent geographical expansion. Diagnosis of the infections caused by these nematodes is indeed challenging. This report describes a novel multiplex PCR able to identify simultaneously three species of lungworms (Aelurostrongylus abstrusus and Troglostrongylus brevior) and heartworms (Angiostrongylus chabaudi) affecting felids. Epidemiological and clinical perspectives are discussed.

TEXT

The so-called cat lungworm Aelurostrongylus abstrusus (Metastrongyloidea, Angiostrongylidae) parasitizes the respiratory system of cats in all corners of the world (1). In the past few years, another respiratory metastrongyloid, Troglostrongylus brevior (Metastrongyloidea, Crenosomatidae), previously described over the last century in wild felids from the Middle East, has been recorded in domestic cats from Mediterranean Europe (1, 2). Recently, the almost unknown heartworm Angiostrongylus chabaudi (Metastrongyloidea, Angiostrongylidae), recorded only in the last century in wildcats until recently (3), has been found in two domestic cats from Italy (4, 5).

The diagnostic stage (i.e., first-stage larvae [L1s]) of A. abstrusus and T. brevior presents overlapping features which render their discrimination very challenging (1); importantly, L1s of A. chabaudi have not been described thus far. It can be argued, however, that they present overlapping features with those of the metastrongyloids A. abstrusus and T. brevior.

Therefore, it is obvious that the occurrence of cardiopulmonary nematodes other than A. abstrusus in domestic cats has been practically ignored until recently. If one considers that felid cardiopulmonary nematodes are regarded as emerging parasites, this lack of knowledge has caused important gaps in feline parasitology.

Hence, in consideration of these new infections in cats, a novel assay for the simultaneous identification and discrimination of A. abstrusus, T. brevior, and A. chabaudi has been validated here. The ribosomal internal transcribed spacer 2 (ITS2) of A. chabaudi was characterized, as previously described (6, 7), using two individual nematodes collected at the necropsy of a domestic cat infected also with A. abstrusus and T. brevior. The alignment of the ITS2 of A. chabaudi with that of A. abstrusus (GenBank accession number DQ372965.2) and T. brevior (GenBank accession number KM506759.1) showed high interspecific differences, which allowed us to design a primer (AChFor: 5′-TCAAAGAAATAATCATCGAAC-3′) specific for the heartworm A. chabaudi (Fig. 1). Therefore, the diagnostic ability of a triplex seminested PCR using primers specific for A. abstrusus (AabFor: 5′-GTAACAACGATATTGGTACTATG-3′) (6), T. brevior (TbrFor: 5′-CGGTGATTGATAATGATGCAT-3′) (7), and A. chabaudi was evaluated here.

FIG 1.

Sequence alignment of internal transcribed spacer 2 plus 5.8S and 18S flanking regions (shaded in gray) of Angiostrongylus chabaudi, Aelurostrongylus abstrusus, and Troglostrongylus brevior, illustrating the nucleotide sequences (in bold italics) targeted by universal primers NC1 and NC2 and by the primer specific for A. chabaudi (AChFor), A. abstrusus (AabFor), and T. brevior (TbrFor). The consensus sequence of A. abstrusus has been obtained based on sequences in GenBank under accession number DQ372965.2. The consensus sequence of T. brevior has been obtained based on sequences in GenBank under accession number KM506759.1.

This novel assay consisted of a first step with universal primers NC1 and NC2 (6, 7) and a second step using primers AabFor, TbrFor, and AChFor in combination with NC2 to achieve specific amplifications of ∼326-bp-, ∼520-bp-, and ∼200-bp-long fragments for A. abstrusus, T. brevior, and A. chabaudi, respectively. The efficiency of this multiplex PCR was confirmed using DNA extracted from individual specimens of A. abstrusus, T. brevior, and A. chabaudi and DNA of each metastrongyloid spiked in a single sample. Moreover, to confirm the laboratory efficiency of this assay, PCRs have been performed on the following samples previously scored positive for lungworms in other studies: (i) DNA extracted from samples obtained in a study carried out in 2007 to 2008 to validate a nested PCR specific only for A. abstrusus (n = 22 fecal samples) (6), (ii) DNA extracted from samples collected from cats diagnosed with aelurostrongylosis that were included in two efficacy studies in 2008 to 2009 (n = 23 fecal samples) (8, 9), and (iii) DNA extracted from cats diagnosed with aelurostrongylosis and/or troglostrongylosis in an epidemiological study carried out in Italy in 2012 to 2015 (n = 84 fecal samples) (10).

Aelurostrongylus abstrusus-positive samples from studies i and ii mentioned above were recently reexamined with a PCR specific only for T. brevior (11).

All amplifications have been carried out using the following protocol. All PCR mixtures were prepared in a 50-μl reaction mixture containing 100 pmol of NC1 and NC2, i.e., primers universal for bursate nematodes, in the first step and 100 pmol of the primers AChFor, AabFor, and TbrFor in the second step, together with 200 pmol of the primer NC2, 4 μl of DNA extract in the first step and 4 μl of template (1:20) in the second step, and 25 μl of AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA), with the addition of distilled water provided by the same manufacturer. PCRs were performed in a thermal cycler (2700; Applied Biosystems, Foster City, CA, USA) using the following cycling protocol: 10 min at 95°C; 40 cycles at 94°C for 1 min, 50°C (first step) or 52°C (second step) for 1 min, and 72°C for 1 min; and a final extension at 72°C for 10 min. Amplicons were electrophoresed in a 1.6% (wt/vol) agarose gel, stained with GelRed 10,000× (Biotium, Inc.). To check for the presence of any contaminations, a negative-control sample containing all of the reaction reagents with sterile distilled water to substitute for the template was added to each PCR run. All the molecular procedures were performed in separate rooms (i.e., DNA preparation and pre-PCR steps in a different room from post-PCR manipulation) to avoid PCR contamination. All procedures were validated twice.

The detection limit of this multiplex PCR was assessed using 10-fold serial dilutions of DNA obtained from single individual specimens of A. abstrusus, T. brevior, and A. chabaudi. The diagnostic sensitivity and possible primer competition of the test were evaluated by spiking these DNA dilutions for each of the individual nematode species.

The specificity of the assay was also assessed. Selected amplicons were purified using a QIAquick gel extraction kit (Qiagen, GmbH, Hilden, Germany) and then sequenced directly using BigDye Terminator v.3.1 chemistry (Applied Biosystems, USA). Sequences were determined in both strands, aligned, and then compared with each other and with those of metastrongyloids available in GenBank using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). The diagnostic specificity of the single-step PCRs for the A. abstrusus and T. brevior assays was previously shown using DNA from other common endoparasites of cats (6, 7). Analogously, the specificity of the present assay was again validated using a panel of DNA samples from other parasites affecting cats (Table 1).

TABLE 1.

DNA samples used to verify the specificity of the multiplex-PCR assay for the simultaneous identification of Aelurostrongylus abstrusus, Troglostrongylus brevior, and Angiostrongylus chabaudi ITS2^a

Sample	Stageb
Cystoisospora felis	O
Cystoisospora rivolta	O
Giardia intestinalis	C
Mesocestoides lineatus	P
Dipylidium caninum	P
Taenia taeniaeformis	P
Ancylostoma tubaeforme	Sa
Uncinaria stenocephala	Sa
Oslerus rostratus	Sa
Toxocara cati	Sa
Toxascaris leonina	Sa
Eucoleus aerophilus	Sa

^aSome samples used were already evaluated in references 6 and 7.

^bO, concentrated oocysts of fecal origin; C, concentrated cysts of fecal origin; P, proglottid from adult specimens; Sa, single adult specimen.

The concordance between the multiplex PCR and the other tests (e.g., single PCRs and Baermann's method) applied to the fecal samples was calculated by Cohen's kappa coefficient (κ) and McNemar's chi-square test, using 2 × 2 contingency tables, where single PCRs and Baermann's method were considered the comparative tests and multiplex PCR was considered the alternative test. The relative concordances of the multiplex PCR with regard to single PCRs and Baerman's method were estimated according to single infections and mixed infections. The κ values obtained were interpreted according to the following guidelines: <0.2 = slight agreement, 0.2 to 0.4 = fair, 0.4 to 0.6 = moderate, 0.6 to 0.8 = substantial, >0.8 = almost perfect, and 1 = perfect agreement (12). Moreover, the rates of animals positive for single or multiple infections by each test were calculated and compared. All statistical analyses were performed using the WINPEPI (PEPI for Windows) epidemiological freeware, and the significance level was set at a P value of ≤0.05.

The results showed that the assay was able to produce single specific amplicons when applied to DNA of individual nematodes and all expected PCR products when mixed DNA was used (Fig. 2). When these PCRs were developed as a single-step PCR for A. abstrusus and T. brevior, amplicons from the first step were not constantly detectable on a gel, while the amplification of the second step was constant for all molecularly positive samples (6, 7). This was the case also with the multiplex PCR presented here.

FIG 2.

Example of an agarose gel showing results by the seminested multiplex PCRs specific for Aelurostrongylus abstrusus, Troglostrongylus brevior, and Angiostrongylus chabaudi. Lane 1: A. abstrusus DNA from an adult stage (second round, primers AabFor-NC2; size, 326 bp). Lane 2: T. brevior DNA from an adult stage (second round, primers TbrFor-NC2; size, 520 bp). Lane 3: A. chabaudi DNA from an adult stage (second round, primers AchFor-NC2; size, 200 bp). Lane 4: A. abstrusus, T. brevior, and A. chabaudi DNA from individual adult stages spiked in a single sample (second round, primers TbrFor, AabFor, AchFor, and NC2). Lane 5: negative control (no-DNA sample). Lane M: size marker.

The sensitivity of the assay proved satisfactory. In fact, when carried out on dilutions of individual DNA samples, the assay was able to detect concentrations as low as 3.1 × 10⁻³ μg/μl, 5.1 × 10⁻³ μg/μl, and 4.8 × 10⁻³ μg/μl for A. abstrusus, T. brevior, and A. chabaudi species, respectively, while it detected concentrations as low as 3.7 × 10⁻³ μg/μl in the spiked samples containing the DNA of the three parasites. None of the DNA samples from other parasites (Table 1) used to verify the specificity of the multiplex PCR assay produced detectable amplicons. When applied to samples collected in previous studies, this molecular assay was able to detect A. abstrusus and T. brevior DNA in samples even when Baermann's method (i.e., the gold standard copromicroscopic technique used to diagnose infections by cardiorespiratory nematodes) was negative (Table 2). All examined samples scored molecularly negative for A. chabaudi (Table 2). Specifically, of the 80 cats positive only for A. abstrusus, all animal samples scored positive by single PCR, multiplex PCR, and Baermann's method. However, of the 34 animals infected only with T. brevior, 33 were positive by single PCR, multiplex PCR, and Baermann's method, while only one Baermann's test-negative cat was positive by both single and multiple PCRs. With regard to cats with a mixed infection by A. abstrusus and T. brevior, a number of different combinations of tests were positive, although 12 of 15 animals showed positivity for all the tests applied (i.e., three cat samples were negative with Baermann's method but positive with single and multiplex PCRs).

TABLE 2.

Comparative performances of different tests applied to 129 fecal samples shown to be positive for cardiopulmonary nematode diagnosis by at least one test

Parameter	Single PCR resulta			Baermann's test result			Multiplex PCR result (organism[s])	Total no. of positive samples
	A. abstrusus	T. brevior	A. chabaudi	A. abstrusus	T. brevior	A. chabaudi
Fecal sample (n = 129)	+	−	NA	+	−	−	+ (A. abstrusus)	80
	−	+	NA	−	+	−	+ (T. brevior)	33
	−	+	NA	−	−	−	+ (T. brevior)	1
	+	+	NA	+	+	−	+ (A. abstrusus/T. brevior)	12
	+	+	NA	+	−	−	+ (A. abstrusus/T. brevior)	2
	+	+	NA	−	+	−	+ (A. abstrusus/T. brevior)	1
Total no. of positive results	95	49	0	94	46	0	80 (A. abstrusus), 34 (T. brevior), 15 (A. abstrusus/T. brevior)	129

^aNA, not available.

The numbers of A. abstrusus infections detected by fecal examination were equivalent when the multiplex PCR (n = 80) and Baermann's test were compared, as reported in Table 3. Similar results were achieved for T. brevior, i.e., 34 of 34 samples scored positive by both single and multiplex PCRs, while Baermann's method exhibited inferior sensitivity in detection of T. brevior compared to A. abstrusus (false negative, 2.95% versus 0%). The number (n = 15 of 15) of mixed infections detected by the single PCRs was the same as that recovered by the multiplex-PCR approach (Table 3); however, a discordance of 3 false-negative results was observed when Baermann's method was applied.

TABLE 3.

Positivity rates for cardiopulmonary nematodes using different diagnostic approaches

Test	No. (%; 95% CI) of positive samplesa
	A. abstrusus (n = 80)	T. brevior (n = 34)	A. chabaudi (n = 0)	A. abstrusus/T. brevior (n = 15)
Single PCR: A. abstrusus	80 (100; 98–100)			15 (100; 98–100)
Single PCR: T. brevior		34 (100; 98–100)		15 (100; 98–100)
Baermann's	80 (100; 98–100)	33 (97.05; 95.3–98.5%)	0 (0)	12 (80; 76.5–85.3)
Multiplex PCR	80 (100; 98–100)	34 (100; 98–100)	0 (0)	15 (100; 98–100)

^aTotal number of cats = 129; CI, confidence interval.

A comparison of the results obtained by Baermann's method and single PCRs examined for concordance with results of the multiplex PCR is presented in Table 4. The overall agreement exhibited by multiplex PCR versus single PCR was highly reliable (κ = 1), as were the relative agreements with respect to single infections and double infections with T. brevior and A. abstrusus.

TABLE 4.

Comparison of multiplex PCR, single PCRs, and Baermann's method in the diagnosis of cardiopulmonary nematodes in 129 cats^a

Test	Multiplex PCR results (no. of samples)		McNemar's test (P)	Cohen's κ	% positive agreement (95% CI)	% negative agreement (95% CI)	% overall agreement (95% CI)
	+	−
Single PCR on feces
Single infection: T. brevior
Positive	34	0	0.5	1	100	100	100
Negative	0	80
Single infection: A. abstrusus
Positive	80	0	0.5	1	100	100	100
Negative	0	34
Double infection: T. brevior + A. abstrusus
Positive	15	0	0.5	1	100	100	100
Negative	0	114
Baermann's
Single infection: T. brevior
Positive	33	0	0.15	0.98	98.5 (95.6–100.0)	99.4 (98.2–100.0)	99.1 (98–100)
Negative	1	80
Single infection: A. abstrusus
Positive	80	0	0.15	0.98	99.4 (98.2–100.0)	98.6 (95.7–100)	99.1 (98–100)
Negative	1	34
Double infection: T. brevior + A. abstrusus
Positive	12	0	0.042a	0.88	88.9 (71.8–100)	98.7 (96.6–100)	97.7 (95.2–98)
Negative	3	114

^aP < 0.05 is statistically significant.

The overall agreement was considered optimal also between Baermann's method and the multiplex PCR, despite the one additional sample positive for T. brevior that was detected only by the multiplex PCR. Nonetheless, the relative agreement between multiplex PCR and Baermann's method was a little bit lower (κ = 0.88) when mixed infections were examined. In particular, McNemar's chi-square test evidenced the difference in the proportions of T. brevior-plus-A. abstrusus positivity between the two different diagnostic approaches (P = 0.042), as the multiplex PCR performed on fecal samples was able to detect three additional positive samples compared to the copromicroscopic technique; this means that Baermann's method has a lower performance value when used in animals with mixed infections (Table 4).

This multiplex PCR test proved to be more accurate than copromicroscopy; however, false-negative results cannot ultimately be ruled out. Nonetheless, this verification could be performed only by a comparison of molecular results with necropsy of study cats. For obvious reasons, this approach is unfeasible. In any case, the dilution tests and the lower DNA threshold detectable by this PCR make the possibility of false-negative results very low.

The lack of samples positive for the rare species A. chabaudi in the study samples and the limited number of sequences available make worthwhile further studies to investigate a possible heterogeneity in the population at the primer-binding regions that leads to false-negative results. Unfortunately, at the moment, this appears highly problematic for the very few A. chabaudi specimens recovered so far. In any case, the ITS2 regions of A. chabaudi here generated were identical to each other and displayed 100% homology with sequences from a European wildcat from Germany (GenBank accession number KM216825.1) and 99% identity with A. chabaudi isolate N.1 from Sardinia, Italy (GenBank accession number KM009115.1).

In conclusion, the results presented here show that multiplex PCR has several advantages compared with classical fecal examinations and monospecific PCR. From an epidemiological point of view, it is worth noting that no sample positive for metastrongyloid larvae scored molecularly positive for A. chabaudi. Although more studies are required to confirm the efficiency of this PCR in field samples collected from cats with unknown parasitological statuses, this method is a promising basis for further studies aiming at filling crucial gaps in the knowledge about cat lungworms and heartworms, e.g., occurrence of these neglected infections in cat populations, efficacy of parasiticides, investigation of the factual role of A. chabaudi in cardiorespiratory diseases of cats, and the role of different mollusk species as intermediate hosts.