Occurrence of Dirofilaria immitis infection in shelter cats in the lower Rio Grande Valley region in South Texas, United States, using integrated diagnostic approaches

Abstract

Dirofilaria immitis is a mosquito-borne filarioid nematode that affects dogs and cats. Although heartworm infections in cats can be fatal, it is commonly neglected by cat owners and veterinarians. Moreover, diagnosing heartworm infection in cats can be challenging, requiring the integration of multiple laboratorial tests and clinical examination. The objective of this study was to estimate the occurrence of D. immitis infection in shelter cats in the Lower Rio Grande Valley (RGV) region of Texas using a combination of immunodiagnostic and molecular methods. The RGV has a large population of stray animals with limited access to veterinary care.

One hundred and twenty-two paired samples of serum and DNA extracted from the blood clots of cats from 14 towns in this region were analyzed. Serum samples were used for heartworm antibody detection (Heska^® Solo Step^®), and heartworm antigen detection using a commercial ELISA kit (DiroCHEK^®) pre- and post-immune-complex dissociation (ICD) via heat treatment. A species-specific probe-based qPCR assay targeting a fragment of the cytochrome oxidase c subunit 1 of the mitochondrial DNA was utilized to detect the presence of parasite DNA. Twenty-two cats (18%) were positive in at least one diagnostic test. Antibody testing detected most cases (19/122; 15.6%); pre- and post-ICD antigen testing detected 6 cases (6/122; 4.9%); and qPCR detected the fewest cases (4/122; 3.3%), with 2 cats positive on all three diagnostic tests. Veterinarians should encourage local cat owners to utilize year-round heartworm prevention.

1. Introduction

Dirofilaria immitis is a filarioid nematode that infects companion animals such as dogs, cats, and ferrets, causing heartworm disease. Compared to dogs, detecting the heartworm infection in cats is challenging for veterinarians due to the greater occurrence of asymptomatic cases, the difficulty of diagnosis, and the lack of approved adulticidal pharmaceuticals (Lee and Atkins, 2010). For these reasons, the American Heartworm Society recommends using integrated diagnostic techniques to detect heartworm infections in cats. (American Heartworm Society, 2020).

Cats, similar to other susceptible definitive hosts, become infected with heartworms when an infected mosquito takes a blood meal and transmits the infective third-stage larvae (L3). After months of migration, D. immitis larval stages mature to adults, which commonly reside in the heart and pulmonary arteries. Unlike dogs, which can harbor 30 or more adult worms in their heart and lungs, infected cats usually have 6 or fewer adult worms, and single-sex D. immitis infection is common (Lee and Atkins, 2010). Eventually the adults will mate and produce microfilariae which circulate throughout the bloodstream (Hays et al., 2020). Although many infected cats remain asymptomatic, clinical presentations of disease can occur much earlier in cats than dogs, with a spectrum ranging from very subtle to severe (Litster and Atwell, 2008; Winter et al., 2017). Clinical signs may include respiratory and neurological disorders, as well as gait alterations, periodic vomiting, lack of appetite, weight loss, ascites, or sudden death. Infection with D. immitis does not always produce patent adult infections in cats. However, cats can develop severe respiratory disease, heartworm-associated respiratory disease (HARD), in response to larval heartworm infections (Winter et al., 2017).

In 2017, a large national effort with unprecedented sample sizes of client-owned cats (over 100,000 cats tested) provided a contemporary estimate of the prevalence of anti-D. immitis antibodies and antigens, with estimates of 1.19% and 0.68% of cats in the United States, respectively. Data estimate from the state of Texas, the presence of anti-D. immitis antibodies and antigens was estimated at 1.32% and 2.11%, respectively (Companion Animal Parasite Council, 2023). Texas is considered an endemic region for heartworm disease due to the ideal climate to support vector populations. The prevalence of heartworm infection in cats is usually assumed to be about 5–10% of the prevalence in dogs in a given area (Litster et al., 2008), but a more recent study challenges this assumption (Hays et al., 2020).

The lower propensity of cats to have adult heartworms, and the late detection of antigens, between 6 and 9 months of age, require the integration of a wider range of diagnostic tools to detect the infection in felines (American Heartworm Society, 2020). In this case, the cost of the recommended association of screening tests for the detection of antigens and antibodies with imaging tests and with the evaluation of other blood parameters can make the diagnosis and treatment of infected cats unfeasible, making chemoprophylaxis with macrocyclic lactone-based products the main determinant of infection control in felines (Lee and Atkins, 2010).

The objective of this study was to determine the occurrence of heartworm infection amongst shelter cats in South Texas. Assessing heartworm exposure and infection in shelter cats may inform on risk for pet cats that are not on compliant prevention in a given area. The southern United States, including Texas, is recognized as an endemic region for heartworm infection (Self et al., 2019). This area is an impoverished community with limited access to veterinary public health efforts.

2. Materials and methods

2.1. Study location

This study was conducted at a large animal shelter located in the Lower Rio Grande Valley region, southwestern region of Texas, USA, along the border with Mexico (26° 13′ 12” N, 98° 7′ 12”W), an endemic region for several vector-borne diseases including heartworm disease (Scavo et al., 2022). The shelter in Hidalgo County intakes approximately 33,000 animals annually and an annual average of 9000 domestic cats (Zecca et al., 2020). These cats are predominately stray cats that are dropped off by animal control agencies; some are surrendered by their owners or dropped off by community members. Nationwide and in this region, surveillance for heartworm infection in cats is poor (Companion Animal Parasite Council, 2023).

2.2. Sample collection

Blood samples (n = 122) were collected in the winter, spring, and summer of 2017. Necropsy was also performed on all cats represented in this study. However, the cats were euthanized for reasons unrelated to this study. Serum was isolated via centrifugation at 5488 ×g for 8 min. DNA was extracted from the blood clot and tissues using the E.Z. Tissue DNA Kit according to the manufacturer’s instructions as described previously (Zecca et al., 2020). All serum samples were stored at −80 °C. As inclusion criteria, only adult cats were included, regardless of coat, sex, or age. Kittens were distinguished from adult cats based on the presence or absence of deciduous canines. Demographic and epidemiological information was collected including sex, size, season of sampling, intake reason, and jurisdiction of origin. Size was determined by shelter staff using the following weight criterion: small (<2.7 kg), medium (2.7–4 kg), and large (>4 kg) (Zecca et al., 2020).

2.3. Diagnostic testing

In total three diagnostic tests were performed: antigen detection, antibody detection, and microfilaria DNA detection. A modified Knotts test for microfilariae detection was not performed due to the samples being frozen for a prolonged period.

2.3.1. Antigen testing

The serum samples were tested for D. immitis antigen using a commercial qualitative enzyme-linked immunosorbent assay (ELISA) (DiroCHEK^® Heartworm Antigen Test; Zoetis, Parsippany, NJ, USA) according to the manufacturer’s instructions. This kit is validated for use in both dogs and cats. Each sample was processed in two manners: pre- and post-immune complex dissociation (ICD) via heat treatment, followed by an optical density reading (O.D.) (Little et al., 2014). A positive and negative control were included in each plate. Samples were considered positive if there was a visible color change after the suggested incubation time and if the O.D. reading was comparable to the O.D. for the positive control. Samples that did not meet the criteria for a positive test were considered negative for D. immitis antigen detection. The O.D. was measured using a spectrometer (Epoch, BioTek Instruments Inc., Winooski, VT, USA) at 590 nm (Sobotyk et al., 2022). The DiroCHEK^® Heartworm antigen test had a sensitivity of 86.2% and a specificity of 99.1% (Little et al., 2014).

2.3.2. Antibody testing

Aliquots of 500 μL of serum from each sample were submitted to the Heska Diagnostic Laboratory for testing for antibody detection (Heska^® Solo Step^® Feline Heartworm Test; Heska, Loveland,CO, USA). An antibody titer of 5 AbU/mL or higher was considered a positive result (Heska, Loveland, CO). A previous study found that this commercial antibody-detection test had a sensitivity of 89.5% and a specificity of 77.5% for serum samples with >4 AbU/mL and a sensitivity of 57.9% and a specificity of 94.2% for serum samples with >19 AbU/mL (Snyder et al., 2000).

2.3.3. qPCR for detection of microfilariae DNA

All samples were tested for the presence of D. immitis microfilariae DNA by simplex probe-based TaqMan real-time PCR (qPCR) as described in Negron et al. (2022) and Sobotyk et al. (2022). The cytochrome c oxidase subunit 1 (cox1) of the mitochondrial DNA was amplified using the forward primer Fil.COI.749-F (5′- CAT CCT GAG GTT TAT GTT ATT ATT TT-3′) and reverse primer Fil.COI.914-R (5′- CWG TAT ACA TAT GAT GRC CYC A-3′). A Taqman probe specific to D. immitis, D.imm. COI.777-P (6FAM-CGG TGT TTG GGA TTG TTA GTG-TAMRA) was also used in the reaction. The final volume of the reaction mixture was 20 ul which included 5 μL of the DNA template, 10 μL (2×) of the Taqman Multiplex Master Mix (Applied Biosystems, Waltham, MA), 50 μM of each primer, and 20 μM of the probe. The reaction was performed in a QuantStudio 3 Real-Time PCR System (Thermo Fischer Scientific, USA). The cycling conditions followed Negron et al. (2022). Each qPCR plate contained a negative and positive control. The positive control was DNA extracted from adult D. immitis and the negative control was nuclease-free water.

2.4. Epidemiological analysis

Heartworm infection occurrence was calculated as the number of cats that had a positive result on one or more tests divided by the total number of cats enrolled. Pearson’s Chi-square test and Fisher’s exact test were used to determine differences between proportions and means. To identify risk factors for the occurrence of D. immitis infection and its association with exposure variable, bivariate analysis and logistic regression were performed to calculate odds ratios and adjusted odds ratios (OR). Statistical analysis was performed using Stata/BE version 17 (StataCorp. 2021. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC).

2.5. Parasite sequencing

All samples that had a positive result on antibody, antigen, and/or qPCR microfilariae detection were subjected to Sanger Sequencing. The cytochrome oxidase c subunit 1 of the mitochondrial DNA (cox1) gene was amplified using COIint forward (5′-TGA TTG GTG GTT TTG GTA A-3′) and COIint reverse (5′-ATA AGT ACG AGT ATC AAT ATC-3′) primers (Casiraghi et al., 2001). A PCR assay was utilized with a 25 μL final concentration: 0.625 μL of 10 μM forward primer, 0.625 μL of 10 μM reverse primer,12.5 μL of 2× GoTaq Green (ProMega Corporation, Madison, Wisconsin, USA), and 2.5 μL DNA template. The cycling conditions included a denaturation step at 95 °C for 2 min followed by 40 cycles at 95 °C for 45 s, 52 °C for 45 s, 72 °C for 90 s, and an extension step at 72 °C for 5 min. A 1% agarose gel stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA) was ran using the PCR products. Setaria equina was used as the positive control and nuclease-free molecular grade water was used for the negative control. All samples that had a band were subjected to PCR purification using the E.Z.N.A.^® Cycle Pure Kit (Omega Bio-Tek Inc., Norcross, GA, USA) per the manufacturer’s instructions, molecular-grade water was used in the wash and elution steps. These samples were sent for Sanger sequencing at Eurofins, and sequencing results were analyzed using 4Peaks (RRID: SCR_000015) and Geneious Prime. Sequences were aligned and compared to a homologous sequence of D. immitis from dogs in Texas (0P681145) (Scavo et al., 2022) prior to submission to GenBank.

3. Results

3.1. Demographic results

Of the 122 cats, the majority were strays (n = 106) with the remainder being surrendered by their owner (n = 14) or a public drop-off (n = 2). The sex ratio was nearly equal, with 54.1% female (66/122). Winter was the largest sampling period (n = 77) followed by spring (n = 29). Summer was the smallest sampling period (n = 16). Most cats were categorized as large (n = 67) followed by medium (n = 50) and small (n = 5).

Thirteen municipalities were represented, McAllen, Pharr, Edinburg, San Juan, Alamo, Alton, Palmview, Monte Alto, Elsa, Weslaco, Donna, Harlingen, and Mercedes. The majority of the cats were from McAllen, TX (43/122; 35.2%).

3.2. Diagnostic test results

Heartworm antigen testing yielded an occurrence of 4.9% (6/122) in this population. All the cats that were heartworm antigen positive had a positive pre and post – ICD result. The post-ICD samples tended to have higher O.D. values and a deeper blue color than the pre-ICD samples. O.D. readings for pre-ICD samples ranged from 0.042 to 0.838. O.D. readings for post-ICD samples ranged from 0.043 to 0.749.

Heartworm antibody testing identified more positive cases and yielded an occurrence of 15.6% (19/122) in this population. About 63% (12/19) of the antibody positive cats in this study had low antibody titers (5–19 AbU/mL) which was indicative of either an exposure to heartworm or current infections with larval stages. The remaining antibody positive cats (7/19) had antibody titers consistent with an active adult heartworm infection (20–61 AbU/mL).

There were three cats that were heartworm antigen and heartworm antibody positive. These cats tended to have lower antibody titers (6–13 AbU/mL) and higher pre and post ICD O.D. readings than other cats that were positive on only one diagnostic test.

Microfilariae detection via qPCR identified the fewest positive cases and yielded an occurrence of 3.3% (4/122) in this population. The cycle threshold (CT) values ranged from 26.2 to 37.9. All four of the cats that had a positive qPCR test also had a positive antigen test which confirmed the presence of an active, adult heartworm infection in these cats.

There were five cats that were positive on at least two diagnostic tests. Of these, two were positive on all three diagnostic tests. Cats that were positive on two or more tests tended to have higher O.D. readings than cats that were positive on only one test. Antibody titers for these five cats ranged from 6 AbU/mL to 25 AbU/mL. All the cats that were qPCR positive also had another positive diagnostic test result (Table 1).

Table 1.

Comparison of qPCR, antibody, and antigen test results for 22 D. immitis positive cats and optical density readings.

Sample ID	qPCR		Conventional PCR	Feline Solo Step Antibody		DiroCHEK Antigen		O.D. Readings
	Result	CT value	Result	Result	Titer (AbU/mL)	Pre-ICD	Post-ICD	Pre-ICD	Post-ICD	−control	+ control
30	−		−	+	5	−	−	0.047	0.043	0.043	0.162
34	−		−	+	31	−	−	0.049	0.067	0.048	0.338
40	−		+	+	61	−	−	0.059	0.064	0.048	0.338
42	−		−	+	18	−	−	0.071	0.046	0.048	0.338
52	−		−	+	8	−	−	0.044	0.043	0.043	0.371
66	−		−	+	22	−	−	0.043	0.044	0.043	0.371
67	−		−	+	28	−	−	0.044	0.045	0.043	0.371
68	−		−	+	9	−	−	0.048	0.044	0.043	0.371
87	−		−	+	6	+	+	0.749	0.659	0.044	0.492
93	−		−	+	12	−	−	0.042	0.043	0.044	0.492
96	−		−	−		+	+	0.139	0.481	0.044	0.492
100	−		−	+	19	−	−	0.044	0.046	0.043	0.494
111	+	35.3	+*	+	11	+	+	0.514	0.456	0.043	0.494
113	+	29.3	+	+	13	+	+	0.177	0.609	0.043	0.494
117	−		−	+	5	−	−	0.046	0.046	0.043	0.494
118	−		−	+	5	−	−	0.044	0.043	0.043	0.494
139	−		−	+	20	−	−	0.045	0.044	0.045	0.542
140	−		+	+	41	−	−	0.048	0.045	0.045	0.542
141	+	26.2	+*	−		+	+	0.237	0.309	0.045	0.542
144	−		−	+	6	−	−	0.044	0.044	0.045	0.542
154	−		−	+	25	−	−	0.047	0.046	0.045	0.542
167	+	37.9	+	−		+	+	0.838	0.789	0.045	0.542

^*Indicates a sequence which was submitted to GenBank.

Combining the results of all diagnostic tests, the occurrence of D. immitis infection/exposure in this population was 18% (22/122).

Most of the positive cases were from stray cats (n = 19), cats sampled during the winter (n = 16), and cats categorized as large (n = 13). There were an equal number of positive cases between males and females (n = 11). None of the positive cats were public drop offs but three cases were from owner surrendered cats. Most of the positive cases were from cats from McAllen, TX (7/22; 31.8%). The cities of Harlingen, Weslaco, Donna, Mercedes, and Elsa did not have any positive cases.

3.3. Epidemiological analysis results

Bivariate analysis was conducted to determine the relationship between D. immitis infection status and different demographic factors. The demographic factors evaluated included sex, size, intake reason, jurisdiction, euthanasia reason, and sampling season. There was no association between any of the demographic factors and D. immitis infection status. Due to there being no significant associations upon bivariate analysis, model building via logistic regression did not occur (Table 2).

Table 2.

Bi-variate analysis of demographic factors associated with heartworm infection/ exposure (n = 122).

Variable	Category	N	Infected (%)	p-value	OR	95% CI
Sex	Male	56	11 (19.6%)	rc
	Female	66	11 (16.7%)	0.67	0.82	0.32–2.06
Size	Small	5	1 (20%)	rc
	Medium	50	8 (16%)	0.82	0.76	0.075–7.74
	Large	67	13 (19.4%)	0.97	0.96	0.099–9.35
Intake	Surrender or Public Drop - Off	16	3 (18.8%)	rc
	Stray	106	19 (17.9%)	0.94	0.94	0.245–3.65
Jurisdiction	McAllen	43	7 (16.3%)	rc
	Pharr	23	3 (13%)	0.73	0.77	0.18–3.32
	Edinburg	18	4 (22.2%)	0.58	1.47	0.37–5.81
	San Juan	16	3 (18.8%)	0.82	1.19	0.27–5.29
	Alamo	8	2 (25%)	0.56	1.71	0.29–10.3
	Other*	14	3 (21.4%)	0.66	1.40	0.31–6.36
Season	Winter	77	16 (20.8%)	rc
	Spring	29	4 (13.8%)	0.42	0.61	0.19–2.01
	Summer	16	2 (12.5%)	0.45	0.54	0.11–2.65

rc = reference category.

^*Includes Alton, Palmview, Monte Alto, Elsa, Weslaco, Donna, Harlingen, and Mercedes.

3.4. Parasite sequencing results

Three of the samples that generated bands were also positive on qPCR (Table 1). We successfully sequenced two of the 5 samples that generated bands using gel electrophoresis, cats 111 and 141 (OQ359098–9). Both sequences were confirmed to be D. immitis and had 100% identity to D. immitis from dogs in Texas (0P681145) (Scavo et al., 2022).

4. Discussion

When the results of all diagnostic tests were combined, the occurrence of heartworm infection/exposure was 18% (22/122). Usually, heartworm prevalence amongst cats is assumed to be 5–15% of the prevalence in unprotected dogs in the same region (Ryan and Newcomb, 1995; Litster et al., 2008). This theoretical prevalence was formulated by three primary observations. The first that cats attract mosquitoes less than dogs which makes it less likely for them to be bitten by an infected mosquito (Venco et al., 2011). The second assumption is that the life-span of heartworms in cats is 50% shorter than in dogs (Venco et al., 2011). The final assumption is that cats also seem to be less susceptible to the establishment of heartworm infections. Only 75% of cats inoculated with D. immitis L3 larvae developed an infection which further complicates heartworm prevalence estimation in cats (McCall et al., 1992). Based off the reported heartworm prevalence for dogs in this region in 2017, we would assume that the heartworm occurrence in cats in this region would be 1–3% (Companion Animal Parasite Council, 2023). However, our reported occurrence was several levels of magnitude higher than this estimate. Hodo et al. (2019) reported a heartworm prevalence of 20.9% amongst dogs from the same shelter as the present study sampled from May 2013 to December 2014. This was not significantly different from the occurrence of heartworm infection in cats reported in this study (p-value = 0.7820). While the samples were not sampled during the same time period, this may provide further evidence that heartworm infection prevalence is similar between dogs and cats. Another study performed on stray cats and dogs in Florida reported similar findings (Hays et al., 2020). Hays et al. (2020) also used integrated diagnostic techniques for heartworm detection, like the present study. They reported that the prevalence of heartworm did not differ between cats and dogs when the results of all diagnostic tests were combined.

Heartworm, especially amongst cats, is severely undertested throughout Texas but especially in the Lower RGV. Over the 2012–2017 period, data from the Companion Animal Parasite Council has shown a relatively stable prevalence of heartworm antigen (1.5–2.1% per year with n = 500–700 cats tested annually) and antibody positive (0.9–1.4% per year with n > 8000 cats tested annually) cats in Texas (Companion Animal Parasite Council, 2023). In 2017, 497,634 dogs were tested for heartworm in Texas. In the same year, 3588 cats were antibody tested and 202 cats were antigen tested in Texas (Companion Animal Parasite Council, 2023). In 2017, three out of the four counties in the Lower RGV (i.e., Hidalgo, Starr, and Willacy) did not report any test results for heartworm antibody or antigen tests in feline patients. In 2017, two heartworm antibody tests and 522 antigen tests to cats have been reported from Cameron County. There were no positive heartworm antibody cats but there were 11 positive heartworm antigen tests amongst the cats tested in Cameron County in 2017. In 2017 Hidalgo County tested 13,034 dogs for heartworm and Cameron County tested 5249 dogs for heartworm. Starr and Willacy counties did not report administering any heartworm tests to dogs (CAPC, 2017) (Table 3). This stark contrast between the number of cats versus dogs tested for heartworm highlights the need for increased heartworm surveillance efforts for cats. While there are flaws in heartworm diagnostics for cats, routine testing and surveillance is important for case identification and risk assessment for the area. Despite the current study being retrospective, reported heartworm prevalence for cats has remained relatively constant from 2017 to 2022 (Companion Animal Parasite Council, 2023). Therefore, our findings are relevant in conveying the burden and risk of heartworm infection on cats in the Lower Rio Grande Valley.

Table 3.

Occurrence of heartworm infection detected by serological tests of cats and dogs in counties of the Lower Rio Grande Valley, Texas, in 2017.

County	Heartworm Antigen (Cats)	Heartworm Antibody (Cats)	Heartworm Antigen (Dogs)
Hidalgo	None reported	None reported	9.03% (1177/13,034)
Cameron	0% (0/2)	0% (0/2)	6.95% (265/5249)
Starr	None reported	None reported	None reported
Willacy	None reported	None reported	None reported

The Companion Animal Parasite Council (CAPC) reports heartworm cases from veterinary clinics which implies that they mainly represent heartworm prevalence in client-owned animals. However, the prevalence reported by CAPC, and the occurrence reported in this study may be comparable due to the underutilization of heartworm prevention. A study conducted on cat owners in the United States and Canada found that heartworm prevention was prescribed for 12.6% of cat owners and that generally preventive use was higher in areas that had a higher heartworm seroprevalence (Levy et al., 2017). They reported that in the west south central georegion of the US (Texas, Arkansas, Louisiana, and Oklahoma), had the highest heartworm seroprevalence amongst cats (0.8%), it did not have the highest heartworm prevention compliance amongst all regions. Significant associations were found between heartworm seroprevalence and cats having outdoor access, where cats that did have outdoor access were at a higher risk of being heartworm positive. Amongst the unowned cats, stray cats were at the highest risk of heartworm seropositivity when compared to owner-surrendered and feral cats (Levy et al., 2017). Due to the large number of stray cats included in this study, it is likely that most of them never received heartworm prevention. Pet owners may perceive the threat of heartworm infection to be higher in the spring and summer months when mosquito vector populations are the highest. While mosquito vector populations do have seasonality, there is still a risk of acquiring a heartworm infection during all seasons. This is especially true in areas with warmer climates such as Southern Texas where mosquitoes are active year-round (Yee et al., 2012). The American Heartworm Society suggests year-round heartworm prevention for cats, especially ones residing in endemic regions (American Heartworm Society, 2020However, education for the relevance heartworm infection in cats amongst both veterinarians and owner is limited which could contribute to reduced compliance amongst cat owners.

During necropsy, presence of adult heartworms was noted in five cats included in this study. Of those five, four were serologically positive for heartworm and two were molecularly positive for heartworm. There was one cat that was confirmed to have adult heartworms during necropsy but did not have a positive result on any of the diagnostic tests. Since this individual did not test positive on any of the diagnostic tests, it was considered negative for the purposes of this study. This highlights the short comings of heartworm diagnostics in cats. Expectedly, no diagnostic test will have 100% sensitivity and specificity, but there are additional challenges presented by the biology of heartworm infections in cats.

For canine heartworm diagnostics it is recommended that heartworm antigen tests are combined with a microfilariae detection test or molecular detection of DNA of microfilariae circulating in blood (Laidoudi et al., 2020; Negron et al., 2022). This may not be ideal for cats since they tend to have single-sex infections and low worm burdens. In turn, it is common for cats to have microfilaremia below the limit of quantification for qPCR assays; thus, resulting in false negatives (Hays et al., 2020). Hence, the low number and the relatively late CT values of qPCR positive cats in this study was anticipated. The primers and probe used in this protocol have shown not to cross-react with other filarioid nematodes that may infect dogs, including Dirofilaria repens and A. reconditum (Laidoudi et al., 2020; Negron et al., 2022).

Currently, it is unknown whether the qPCR protocol utilized in this study may cross-react with Dirofilaria striata, which has been seldomly reported in cats in the US. D. striata typically causes subcutaneous nodules in wild felids but infections have been noted to occur in domestic cats (Wyatt et al., 2020). A case report on D. striata infection in a domestic cat reported that serology did not detect D. immitis antigen (Wyatt et al., 2020) on the WITNESS Canine Heartworm antigen test and the SNAP Feline Triple Test. However, serology was positive for D. immitis antibody using the same diagnostic test as the present study, Solo Step Feline Heartworm Immunoassay (Wyatt et al., 2020). Which may suggest cross-reactivity of D. striata with D. immitis antibody detection assays.

Out of the five samples that produced bands on conventional PCR, three of those were also positive on qPCR. The two that were successfully sequenced had more apparent bands than the other three samples and relatively lower CT values than the other qPCR samples (Table 1).

Antigen testing is considered the ‘gold standard’ for heartworm diagnostics in dogs but can present issues when utilized in cats (American Heartworm Society, 2020). Cats tend to have low levels of circulating heartworm antigen due to low worm burdens and single-sex male infections (Berdoulay et al., 2004) which is further compounded by the formation of heartworm antigen-antibody immune complexes (Little et al., 2018). These two factors can cause false negative results in infected cats. To decrease the chance of false negatives, ICD via heat treatment was performed (Little et al., 2014). When the samples are heated the bonds between the antigen and antibodies break and the antigens can bind to the ELISA plate thus generating a positive result in infected cats (Beall et al., 2017). Pre- and post-ICD samples were tested for heartworm antigen because oftentimes ICD can decrease the specificity of diagnostic tests and increase the chance of cross-reactivity (Szatmári et al., 2020; Venco et al., 2017). Other studies have utilized ICD methods to produce positive antigen test results in heartworm positive cats (Gruntmeir et al., 2017). Gruntmeir et al. (2017) found that when feline serum samples were heat treated, detection of heartworm antigen increased. In our study, however, all cats that tested antigen positive pre-ICD also tested positive post-ICD; therefore, not influencing the occurrence of heartworm in the study population. This may be because all the heartworm positive cats in this study had sufficient levels of heartworm antigen, so heat treatment was not necessary to produce a positive result.

Heartworm antibody testing is also a useful diagnostic technique because it can provide early detection for positive cats. Heartworm antibody tests produce positive results in about 3–6 months because they respond to antibodies created against the larval stages of D. immitis. Unlike dogs which are typically clinically affected by the adult stages of heartworm, larval stages can produce respiratory disease in cats (HARD) (Winter et al., 2017). While antibody testing may seem to be the most reliable and valuable form of testing for heartworm infection in cats it is not always indicative of an active infection. For the heartworm antibody test used in this study antibody titers around 5 AbU/mL were indicative of exposure and antibody titers >19 AbU/mL are indicative of an active infection. In this study, cats that had negative antigen tests tended to have lower antibody titers which indicated that they were either exposed to heartworm or had a larval infection but not an adult heartworm infection. About 80% of asymptomatic cats that are infected with heartworm can self-clear heartworm infections (Lee and Atkins, 2010). It is estimated that 10–20% of cats that test positive on heartworm antibody tests have a mature heartworm infection (Miller et al., 1998). Due to the issues surrounding heartworm diagnostics in cats, it is likely that there were false positives or false negatives which could affect the prevalence estimation.

One limitation of this study is that only shelter cats were included. Since most of the cats were strays, it is likely that they were never exposed to heartworm prevention in the past. Also, since they live outdoors it is likely that they had more contact with vectors than client-owned animals typically would. Therefore, these results cannot be extrapolated to client-owned animals. However, heartworm prevention is underutilized amongst cat owners so the prevalence in client-owned and stray cat populations may be comparable.

5. Conclusion

The high occurrence of heartworm infection and exposure in cats of the lower RGV, south Texas, emphasizes the need for continued education of clients regarding heartworm infection and disease in cats. Veterinarians should encourage year-round heartworm prevention for cats, and proactively utilize integrated diagnostic tests for patient screening.