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. 2026 Jul 8;21(7):e0351437. doi: 10.1371/journal.pone.0351437

Zoonotic endoparasites and Toxoplasma gondii seropositivity in free-roaming cats (Felis catus) from New York City boroughs

Viet-Linh Nguyen 1, Elizabeth Gurtowski 2, Jiayi Chen 2, Megan Rosen 2, Pratap Kafle 1,2,*
Editor: Balbir B Singh3
PMCID: PMC13345238  PMID: 42418442

Abstract

Free-roaming cats (Felis catus) can serve as reservoirs of various zoonotic parasites in urban settings. Despite a large population of free-roaming cats around New York City, studies assessing the prevalence and shedding of various parasites in the New York urban landscape are scarce. This study utilized fecal and blood samples opportunistically collected during the Trap Neuter Return (TNR) program from 87 free-roaming cats in New York City between May and July 2023. Samples were analyzed using centrifugal fecal flotation, coproantigen immunoassays, serologic assays, and PCR-based assays for gastrointestinal and vector-borne parasites. Fecal flotation (n = 87) results revealed that 57.5% (50/87; 95% CI: 46.9–67.4) of cats were infected with at least one species of parasite. The most prevalent infection was Toxocara spp. (54%; 95% CI: 43.4–64.3), followed by Ancylostoma spp. (13.8%; 95% CI: 8.2–22.6) and coccidia (11.5%; 95% CI: 6.4–19.9). Coproantigen testing (n = 43) identified Giardia spp. in 11.6% (5/43; 95% CI: 5.1–24.5) and Cryptosporidium spp. in 2.3% (1/43; 95% CI: 0.4–12.1) of cats. Antibodies to Toxoplasma gondii were detected in 8.9% (4/45; 95% CI: 3.5–20.7) of serum samples; no Dirofilaria immitis antigen and Cytauxzoon felis DNA were found in the blood samples (n = 45). Male cats were significantly more likely to be infected with Toxocara spp. (OR = 4.36) and, along with juvenile cats (<1 year), shed significantly higher numbers of eggs (p < 0.05), identifying young males as high-intensity “super-shedders” driving environmental contamination. The high prevalence of zoonotic helminths, particularly Toxocara spp., underscores the public health risks associated with unmanaged feline populations in densely populated urban centers. These findings highlight the utility of integrating disease surveillance into TNR programs to monitor urban ecosystem health and mitigate zoonotic risks.

Introduction

Free-roaming cats (Felis catus), including stray, feral, and outdoor-access owned cats, are abundant in many densely populated urban environments, where they occupy shared spaces with people, pets, and synanthropic wildlife, increasing the likelihood of environmental exposure to various parasites and pathogens through contaminated soil and public areas. These cats can serve as significant reservoirs for a range of helminths and protozoal parasites, such as Toxocara spp., Ancylostoma spp., Giardia spp., Cryptosporidium spp., and Toxoplasma gondii, that can cause diseases in companion animals and humans, with children and immunocompromised individuals at particular risk [1–5]. From a One Health perspective, unmanaged free-roaming cat populations occupy a critical interface between animal health, human health, and environmental hygiene. Therefore, surveillance of parasitic infections in urban free-roaming cat populations represents an important component of One Health approaches aimed at understanding and mitigating zoonotic risks at the human–animal–environment interface.

Environmental persistence of parasite stages shed by cats further amplifies their public health significance. For instance, eggs of Toxocara spp. are highly resistant to environmental degradation and can remain infective in soil for prolonged periods, leading to widespread contamination of public spaces, indicating potential exposure for people and other animals [6]. Similarly, T. gondii oocysts shed by infected cats can persist in soil and water, contaminate produce, and infect terrestrial and aquatic wildlife, illustrating the cross-ecosystem nature of this parasite [7–9]. Giardia and Cryptosporidium infections in cats involve a mixture of host-adapted and potentially zoonotic assemblages and species, and while the overall zoonotic risk from cats is considered low to moderate, their role as sources and sentinels remains relevant in densely populated settings [3].

In many cities, Trap Neuter Return (TNR) programs are used to manage feral cat colonies, and they offer an efficient opportunity to integrate disease surveillance into ongoing population control and welfare interventions. Leveraging TNR programs for systematic parasitological monitoring can generate data that are directly relevant to One Health, including estimates of infection prevalence, intensity of environmental shedding, and demographic risk factors within urban cat populations.

The objectives of this study were to estimate the prevalence of key gastrointestinal and zoonotic parasites in free-roaming cats enrolled in a TNR program in NYC and to identify demographic risk factors associated with infection and shedding intensity. These data provide an important baseline for understanding the role of free-roaming cats in environmental contamination and inform One Health strategies to mitigate zoonotic risks in densely populated urban environments.

Materials and methods

Study population and sampling

This cross-sectional study was conducted between May and July 2023 using free-roaming cats (defined here as cats not confined indoors and including feral, stray, and colony-associated individuals) captured through the Long Island University College of Veterinary Medicine TNR program from various locations around NYC (Fig 1). Cats were humanely trapped at multiple sites, and the trapping location was recorded. For each individual cat, the attending veterinarians recorded sex and reproductive status, including pregnancy and lactation. Age estimation was based on physical examination, dentition, and body size.

Fig 1. Sampling locations of free-roaming cats trapped through the Long Island University College of Veterinary Medicine Trap Neuter Return program, New York City, USA, May–July 2023 (n = 87).

Fig 1

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Red points denote trap-site coordinates rounded to two decimal places (~1.1 km grid). The figure was prepared in QGIS 3.34 using public-domain shapefiles.

Following physical examination, each cat received a unique microchip identifier and was surgically sterilized as part of the TNR program. The animals were then released at the site of capture. The study procedures complied with institutional guidelines for animal handling and welfare and were approved by the Institutional Animal Care and Use Committee of Long Island University (protocol # 2023−021).

Laboratory analyses

Fresh fecal samples were collected rectally and processed within 24 hours. Quantitative fecal flotation was performed using the Wisconsin double-centrifugation technique with Sheather’s sugar solution (Jorgensen Laboratories, CO; specific gravity 1.27). Briefly, a precisely weighed fecal sample (~2 g, recorded to 0.01 g) was suspended in tap water, strained through cheesecloth, and centrifuged at 400 × g for 5 minutes to pellet the eggs. The supernatant was decanted, the pellet was resuspended in Sheather’s solution, and the suspension was centrifuged a second time at 400 × g for 5 minutes with a coverslip placed on top of a meniscus formed at the tube rim. The coverslip was transferred to a microscope slide, and the entire coverslip area was systematically examined at 100× and 400 × magnification. Helminth eggs and protozoal oocysts were identified based on morphological characteristics [10]. For each sample, all eggs and oocysts on the entire coverslip were enumerated, and eggs per gram (EPG) were calculated by dividing the total egg count by the recorded weight of the fecal sample in grams. This method is widely accepted as a quantitative fecal test yielding EPG values with sensitivity advantages for low- and moderate-intensity infections [11,12]. Due to limited fecal sample volumes, traditional zinc sulfate (ZnSO4) centrifugal flotation for Giardia cyst detection was omitted; instead, samples were processed for coproantigen testing, which is considered more sensitive for detecting Giardia antigen. Also, the insufficient fecal material precluded the use of fecal sedimentation and the Baermann technique, restricting the parasitological assessment to flotation and antigen-based diagnostics only.

Antigen-based, serologic and molecular testing

Due to sample volume and assay availability, coproantigen and serologic testing were performed on subsets of available samples. For this, fecal, serum, and whole blood samples were submitted to the Cornell University Animal Health Diagnostic Center (AHDC) for further analysis.

Fecal samples (n = 43) were tested for Giardia and Cryptosporidium using ProSpecT™ Giardia Microplate Assay (Thermo Fisher, USA) and ProSpecT™ Cryptosporidium Microplate Assay (Thermo Fisher, USA), respectively. Although these commercial enzyme-linked immunosorbent assays (ELISA) were originally developed for human diagnostics, they have been validated for veterinary species at the Cornell AHDC and are verified in-house for the detection of Giardia and Cryptosporidium in veterinary samples. These assays are widely used for detection of protozoal infections in animals and contaminated environments with higher sensitivity compared with flotation alone [13]. Antigen-positive samples were further submitted to the Cornell AHDC for molecular confirmation using their validated in-house PCR assays.

Serum samples (n = 45) were screened for T. gondii antibodies using the modified agglutination test (MAT), with titers ≥1:25 considered evidence of prior exposure, consistent with previous studies in cats and wildlife [14,15]. A commercial heartworm antigen ELISA was performed by the Cornell AHDC as part of their standard diagnostic service to detect circulating Dirofilaria immitis adult-female antigen.

EDTA whole-blood samples (n = 45) were submitted to the Cornell AHDC for detection of Cytauxzoon felis using their validated in-house real-time PCR diagnostic assay.

Data analysis

Parasite prevalence was calculated separately for each diagnostic method using appropriate denominators: fecal flotation (n = 87), protozoal antigen testing (n = 43), and T. gondii serology (n = 45). Prevalence estimates were reported as percentages with 95% confidence intervals. Associations between host demographic factors such as age (young cats: < 1 year old, comprising kittens and juveniles; adults: ≥ 1 year old), sex, lactation status and parasite infection status were assessed using chi-square tests or Fisher’s exact tests when expected cell counts were less than 5, with statistical significance evaluated at p ≤ 0.05. Odds ratios (OR) with 95% confidence intervals were calculated for significant predictors.

Multivariable logistic regression models were constructed to assess independent effects of age and sex on binary infection outcomes (any parasite infection) while controlling for potential confounding. Model coefficients were exponentiated to yield adjusted odds ratios (aOR) with 95% confidence intervals.

For infection intensity analysis, overdispersion in egg per gram (EPG) count data was assessed by calculating variance-to-mean ratios. Due to substantial overdispersion (variance/mean ratio = 835.7), negative binomial generalized linear models (GLMs) were fitted to model EPG as a function of age and sex. Incidence rate ratios (IRR) with 95% confidence intervals were calculated to quantify the magnitude of associations. Non-parametric Mann-Whitney U tests were used to compare EPG distributions between two groups, and Kruskal-Wallis tests were employed for comparisons across three age categories (kittens, juveniles, and adults). All statistical analyses were performed using R version 4.3.1 [16] with packages including tidyverse, MASS, binom, epitools, and DescTools.

Results

Study population

A total of 87 free-roaming cats were sampled and included in parasitological analysis. Among cats with complete demographic data (n = 59), most were classified as young (<1 year old, n = 43, 72.9%), comprising 5 kittens (<6 months) and 38 juveniles (6–12 months), with 16 adults (>12 months, 27.1%). The population included 19 males and 40 females. Among females with known reproductive status, 11 were pregnant and 7 were lactating at the time of examination.

Parasitological findings

Overall, 57.5% (50/87; 95% CI: 46.9–67.4%) of cats were positive for at least one endoparasite species detected by fecal flotation. Toxocara spp. eggs were the most prevalent, detected in 54.0% (47/87; 95% CI: 43.6–64.1), followed by Ancylostoma spp. eggs in 13.8% (12/87; 95% CI: 8.1–22.6), and coccidia oocysts in 11.5% (10/87; 95% CI: 6.4–19.9) (Table 1).

Table 1. Prevalence of parasites in free-roaming cats in New York City. Prevalence with 95% Confidence Interval (CI) was calculated using total samples tested per method.

Parasites Diagnostic method Positive/ Total Prevalence (%) 95% CI
Helminths
Toxocara spp. Fecal flotation 47/ 87 54.0 43.6–64.1
Ancylostoma spp. Fecal flotation 12/ 87 13.8 8.1–22.6
Protozoa
Coccidia Fecal flotation 10/ 87 11.5 6.4–19.9
Giardia spp. Coproantigen ELISA 5/ 43 11.6 5.1–24.5
Cryptosporidium spp. Coproantigen ELISA 1/ 43 2.3 0.4–12.1
Toxoplasma gondii Serology (MAT) 4/ 45 8.9 3.5–20.7
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Co-infections were frequently observed, with 21.8% (19/87; 95% CI: 14.5–31.6%) of the cats harboring two or more parasite species simultaneously. The most common combination was Toxocara spp. and Ancylostoma spp., which was identified in 11.5% (10/87; 95% CI: 6.4–19.9%) of the screened cats.

Antigen testing of 43 fecal samples identified Giardia in 11.6% (5/43; 95% CI: 5.1–24.5) and Cryptosporidium in 2.3% (1/43; 95% CI: 0.4–12.1), including infections that were not detected by flotation alone. Confirmatory PCR performed by the Cornell AHDC did not detect Giardia or Cryptosporidium DNA in any sample.

Of the 45 serum samples tested, 8.9% (4/45; 95% CI: 3.5–20.7) were seropositive for T. gondii antibodies, indicating prior exposure despite the absence of detectable T. gondii oocysts in fecal flotation. No heartworm antigen was detected in any cat, and real-time PCR did not identify C. felis DNA in any tested blood sample.

Risk factor analysis

Risk factor for infection: Univariable analysis revealed no significant demographic risk factors for the overall presence of parasites. The prevalence of “any parasite” did not differ significantly between young cats (69.8%) and adults (62.5%) (p = 0.60), nor between males (84.2%) and females (60.0%) (p = 0.06). When analyzing specific parasites, male sex was identified as a significant risk factor for Toxocara spp. infection (OR=4.36; 95% CI: 1.10–17.37; p = 0.04). No significant demographic associations were found for Ancylostoma spp. or coccidia. While lactating females showed a higher prevalence of coccidia (27.3%) compared to non-lactating females (3.4%), this difference approached but did not reach statistical significance (OR=10.5; p = 0.056).

Infection intensity: While the prevalence of infection was largely consistent across demographics, the intensity of egg shedding exhibited distinct biological patterns. Toxocara spp. egg counts were highly overdispersed and significantly associated with both age and sex (Table 2). A negative binomial GLM identified young cats as “super-shedders,” with an incidence rate ratio (IRR) of 8.74 (p = 0.002) compared to adults. Males also shed significantly higher numbers of Toxocara eggs than females (IRR = 3.67; p = 0.030). In contrast, shedding intensity for Ancylostoma spp. did not differ significantly by age (Young median: 25.0 EPG vs. Adult median: 94.2 EPG; p = 0.52) or sex (p = 0.69). Similarly, coccidia oocyst counts showed no significant intensity differences between demographic groups (p = 0.29).

Table 2. Associations between host demographic factors and both infection prevalence (risk) and egg shedding intensity (burden) for dominant helminths in free-roaming cats around New York City.

Demographic Group N Toxocara spp. Ancylostoma spp.
Prevalence (%)

(OR; 95% CI)		 Median EPG

(IRR; p-value)		 Prevalence (%)

(OR; 95% CI)		 Median EPG

(p-value)
Age
Young (<1 yr) 43 67.4

(1.6; 0.5–5.2)		 84.2

(8.74; 0.002) 		16.3

(0.8; 0.2–3.8)		 25.0

(0.52)
Adult (≥1 yr) 16 56.2

(Reference)		 10

(Reference)		 18.8

(Reference)		 94.2
Sex
Male 19 84.2

(4.4; 1.1–17.4) 		109.3

(3.67; 0.030) 		26.3

(2.5; 0.6–10.0)		 247.0 (0.69)
Female 40 55.0

(Reference)		 10

(Reference)		 11.6

(Reference)		 25.0
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Note: EPG = Eggs Per Gram of feces. OR = odds ratio from Fisher’s exact test. IRR = incidence rate ratio from negative binomial generalized linear model. Median egg counts were calculated among infected animals only. Significant associations are shown in bold.

Discussion

This study reports a high burden of gastrointestinal parasites in free-roaming cats around NYC and highlights important demographic patterns with direct relevance to zoonotic risk and environmental contamination in urban environments. More than half of the cats examined were infected with at least one endoparasite species, with Toxocara spp. and Ancylostoma spp. occurring at prevalences comparable to or exceeding those reported in other urban cat populations in the northeastern United States [17,18]. These findings reinforce the role of free-roaming cats as reservoirs of environmentally persistent zoonotic helminths in densely populated landscapes.

The high prevalence of Toxocara spp. found in this population is consistent with the parasite’s ubiquity and ability to contaminate the environment, particularly in urban areas where feral cat colonies exist and feces are not promptly removed. This rate exceeds those reported in many other urban centers in developed nations and aligns more closely with prevalences found in developing regions or rural environments [19]. Recent environmental surveillance by Tyungu et al. (2020) detected Toxocara eggs in 38.5% of NYC public playgrounds, with contamination rates reaching 66.7% in the Bronx [17]. Critically, that study identified T. cati as the predominant species in soil samples, rather than the canine species T. canis. Our findings support the idea that the free-roaming cat population is the active biological source of this environmental burden. Because Toxocara eggs are highly robust, remaining infective in soil for years andwithstanding the northeastern winter freeze-thaw cycles, environmental shedding by these cats presents a long-term public health risk.

The public health implications of these findings are particularly salient in a context such as New York City. Despite NYC’s high Human Development Index and substantial healthcare infrastructure, seroprevalence estimates indicate that approximately 5% of the U.S. population carries antibodies to Toxocara, with markedly higher rates among children, non-Hispanic Black populations, and residents of low-income urban neighborhoods [5]. Toxocariasis is recognized by the U.S. Centers for Disease Control and Prevention as one of five neglected parasitic infections of public health concern. Children are at disproportionate risk because of geophagia, hand-to-mouth behavior, and play in soil and sand contaminated by free-roaming-cat feces, exposures that are concentrated in dense urban centers where outdoor space is shared between feral colonies and the public. The substantial playground-soil contamination dominated by T. cati that has been documented in NYC public spaces [17], together with the high Toxocara prevalence and the heavy juvenile-male shedding intensity reported here, suggests transmission risk. These findings support the idea of integrating environmental decontamination, public education, and targeted parasite control into TNR programs as a One Health priority.

By quantifying egg counts alongside prevalence, this study adds an important dimension to understanding transmission risk: infection intensity is highly aggregated, with a subset of cats responsible for a disproportionate share of environmental egg output. Egg shedding varied greatly by age and sex, even though parasites were present in high numbers across all demographic groups. Young cats were classified as “super-shedders,” demonstrating an egg shedding intensity nearly tenfold greater than that of adults (IRR = 8.74; p = 0.002). This pattern is consistent with age-acquired immunity and reduced worm fecundity in older hosts, as described for ascarid infections in both domestic and wild carnivores [10]. Male cats were identified as being four times more likely to be infected with Toxocara spp. (OR=4.36) and showed significantly higher egg-shedding intensity than females (IRR = 3.67; p = 0.030). This male bias has been observed in other mammalian host-parasite systems [20] and is often attributed to the immunosuppressive effects of testosterone or behavioral factors such as larger home ranges that increase exposure to contaminated soil and paratenic hosts [21,22]. From a management standpoint, the emergence of young males as “super-shedders” suggests that targeted interventions in this demographic group could yield outsized reductions in environmental contamination. Administering broad-spectrum anthelmintics to heavily shedding juveniles and young males during TNR procedures may be more efficient, in terms of eggs removed from the environment per treatment, than uniformly treating all cats with low-intensity infections. Operationalizing such targeted deworming will require further work on feasibility, cost, and potential for repeated treatment in colonies, but the principle of focusing on high-shedding individuals is well aligned with modern parasite control strategies. It should be noted, however, that egg-output measures are best interpreted as indices of environmental contamination potential rather than as direct estimates of adult-worm burden, since per-female fecundity in Toxocara cati can exceed 100,000 eggs per day and is modulated by immune status, worm age, and density-dependent effects [23].

The observed prevalence of Ancylostoma spp. in this population has important implications for both animal and human health in New York City. Although A. tubaeforme is the most common feline hookworm, its eggs are morphologically indistinguishable from those of the zoonotic species A. braziliense, which cats can also shed [24]. Without molecular speciation, the precise zoonotic risk associated with these infections cannot be determined; however, the presence of a sizeable hookworm-infected free-roaming cat population in a temperate urban setting is noteworthy given ongoing climate-driven shifts in helminth distributions. Reports of A. braziliense in dogs across a broad swath of the United States, including northern regions, suggest that ecological conditions are becoming increasingly permissive for subtropical hookworms, warranting continued surveillance and molecular characterization of feline hookworms in the Northeast [25,26]. Coccidia oocysts were also detected at a moderate prevalence. Although species-level identification was not pursued, these oocysts are most likely members of the Cystoisospora complex that commonly infects cats and primarily affects young or immunocompromised individuals. While feline coccidia are generally regarded as having limited direct zoonotic relevance, their presence in free-roaming cats is epidemiologically relevant because they can contribute to gastrointestinal disease and poor body condition in kittens. The trend toward higher coccidia prevalence in lactating females further suggests that reproductive and nutritional stress may influence susceptibility or shedding. Together, the hookworm and coccidia findings highlight the need to consider both zoonotic risk and animal welfare when designing parasite control strategies for urban TNR programs.

Giardia and Cryptosporidium infections in this study were detected using coproantigen assays applied to a subset of fecal samples. In field-based studies of small mesocarnivores, particularly when fecal samples are collected rectally during brief handling windows, available sample volume is severely limited, and degradation cannot always be prevented before processing. Under these constraints, coproantigen ELISAs offer practical advantages: they require minimal fecal material, are more sensitive than flotation for detecting low-intensity or intermittent shedding (especially for Giardia), and tolerate suboptimal sample preservation better than microscopy. Conventional flotation, by contrast, requires larger volumes and yields lower sensitivity when oocyst/cyst output is sparse. However, coproantigen assays cannot differentiate zoonotic from host-adapted assemblages, and the absence of PCR amplification likely reflects low parasite loads, intermittent shedding, or inhibitors in field-collected samples rather than true absence. These findings underscore the utility of antigen-based methods for TNR surveillance but highlight the need for future work coupling sensitive antigen detection with genotyping to clarify the specific role of urban cats in Giardia and Cryptosporidium transmission cycles relevant to human health.

The non-detection of C. felis DNA in any cat tested is also noteworthy, given the rapid range expansion of its primary vector, the lone star tick (Amblyomma americanum), into New York State [27]. Populations of A. americanum are now well-established on Long Island and Staten Island and have been detected in the Bronx [28]. The presence of the competent vector without the pathogen suggests that NYC is currently a “receptive” zone for C. felis. The lack of infection in our sample likely reflects the absence of the natural reservoir host, the bobcat (Lynx rufus), from the urban core. However, the risk of introduction remains. The movement of infected domestic cats from endemic regions (e.g., the southern US) could theoretically introduce the pathogen to the local tick population, establishing a novel urban transmission cycle. Our negative findings serve as an important baseline against which future emergence can be measured.

The absence of heartworm antigen should be interpreted with caution. Feline heartworm disease is notoriously difficult to diagnose; cats typically harbor low worm burdens (1–3 worms) and often have single-sex infections that do not produce the antigen detected by commercial assays [29]. Studies utilizing antibody tests often reveal exposure rates significantly higher than antigen prevalence. While our results suggest that heartworm infection is not currently endemic in NYC free-roaming cats, the antigen-only screening approach used here cannot rule out subclinical, single-sex, or pre-patent infection. A further limitation is that we did not perform the modified Knott’s concentration test or an antibody (Ab) ELISA. The Knott’s test was not performed because the small blood volumes obtainable from trapped free-roaming cats had to be prioritized across MAT serology for T. gondii, heartworm antigen testing, and qPCR for Cytauxzoon felis. In addition, feline heartworm infection is overwhelmingly amicrofilaraemic; naturally infected cats rarely produce detectable circulating microfilariae, owing to low and often single-sex worm burdens and active immune-mediated microfilaricidal activity [29], so antigen ELISA is the recommended primary screen in this host. Nevertheless, best practice combines antigen detection, antibody detection, and microscopic blood examination, and we recommend that future surveillance work in the region incorporate the full diagnostic triad whenever blood volume permits.

The seroprevalence of T. gondii (8.9%) in our study was lower than that reported in many other free-roaming cat populations, which can exceed 25% [2,30]. This relatively low rate may reflect an “urban shield” effect, where free-roaming cats in dense cities rely more heavily on anthropogenic food sources (e.g., intentional feeding, restaurant and household waste) than on hunting intermediate hosts like rodents and birds, thereby reducing their trophic exposure to tissue cysts. Seropositivity reflects prior exposure rather than active oocyst shedding; therefore, these findings indicate population-level exposure rather than current environmental contamination risk. Given the high density of cats in the city, even a low shedding rate contributes to a significant cumulative environmental load of oocysts, which can contaminate urban gardens and waterways.

Free-roaming cats are considered sentinels for zoonotic pathogens and environmental contamination in urban settings because they occupy diverse habitats, prey on multiple species, and share public spaces with humans [31,32]. The results from this study suggest that TNR programs should ideally be coupled with parasite control strategies where feasible, although the logistics of treating free-roaming populations remain challenging. Collaboration between veterinarians, public health authorities, municipal park services, and wildlife managers can help map hotspots of contamination, implement targeted interventions, and monitor trends in zoonotic pathogen prevalence and environmental contamination over time.

Acknowledgments

The authors thank Marialouise Burgos and participating veterinary clinicians for their assistance with animal handling and sample collection. The authors also acknowledge the Cornell University Animal Health Diagnostic Center (AHDC) for diagnostic support. The authors additionally thank Maged Hemida, Melody Bauer, Abid Shah, Mani Lejeune, and Holly Ann White for their contributions to sample collection, laboratory analysis, and logistical support.

Data Availability

All de-identified data and analysis code required to reproduce the results are publicly available at Zenodo (https://doi.org/10.5281/zenodo.19866812).

Funding Statement

This work was funded by institutional startup funds from Long Island University, College of Veterinary Medicine and Rowan University, Shreiber School of Veterinary Medicine.

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Decision Letter 0

Balbir B Singh

24 Apr 2026

-->PONE-D-26-00846-->-->Zoonotic endoparasites and Toxoplasma gondii   seropositivity in free-roaming cats (Felis catus  ) from an urban environment-->-->PLOS One

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Dear Authors,

Your manuscript entitled “Zoonotic endoparasites and Toxoplasma gondii seropositivity in free-roaming cats (Felis catus) from an urban environment” is a valuable contribution to the necessary surveillance of parasitic infections in animals living in urban settings. Studies of this type should be conducted regularly in order to accumulate important epidemiological data.

I believe that your manuscript is worthy of publication after addressing the following points:

1. Unfortunately, the reference to EPG measurement is not appropriate, as no quantitative method (e.g., McMaster) was applied. I understand that an estimation of the relative parasite burden was performed by counting parasite eggs across the entire preparation; however, this is not sufficient to be expressed using the strictly quantitative EPG metric. Therefore, I would recommend removing the EPG measure throughout the manuscript and, to utilise your approximate estimate of parasite burden, adopting an ad hoc metric, such as “number of eggs per preparation.”

2. I would suggest adding the study area to the title, namely, New York City. This is simply a suggestion, and I leave it to your judgment and preference.

3. Line 118. Please change “Occult Heartworm ELISA test was performed…” to “An ELISA test was performed…”. Note that “occult” refers to a heartworm infection in which microfilariae are not detected in the blood (not investigated in the present study).

4. Line 216. Please change “variant” to “species”.

5. Line 294. Please change “While our results suggest that patent adult heartworm infection is not currently hyper-endemic in NYC feral cats” to “While our results suggest that heartworm infection is not currently endemic in NYC feral cats.”

6. Please add to the Discussion the limitation of not performing the Knott’s test in the investigation of heartworm infection. Although heartworm infection in cats is typically amicrofilaraemic (i.e., occult), microfilaraemia does occur. Given that, a) the number of heartworms in cats is usually very low, b) that a single mature pair of adult heartworms can produce microfilariae, but c) would most probably result in a negative serological test (too little antigen to be detected), laboratory diagnosis in cats should include all possible tests, i.e. the Knott test, Ag detection and Ab detection.

Reviewer #2: The Manuscript Number PONE-D-26-00846, entitled “Zoonotic endoparasites and Toxoplasma gondii seropositivity in free-roaming cats (Felis catus) from an urban environment” show interesting and relevant results on zoonotic endoparasites in New York, particularly a high number of infected animals by Toxocara cati. The results, considering the results, reinforce the strategies employed to deal with parasites of free-roaming cats. However, authors are recommended to carefully revise and to include some information.

I hereby would like to present some recommendations for improvement of the current version of the manuscript.

Major comments

## Materials and Methods

Please, give some details regard to the following procedures:

- kits used for extracting genetic material from faeces.

- Describe the primers adopted in the trials (even in a supplementary file).

- Describe, briefly, the ELISA test protocol for detection of Occult.

## Discussion

Despite the main aim of the researchers was to diagnostic zoonotic endoparasites in free-roaming cats, I suggest the inclusion of a paragraph/sentence concerning human toxocariasis in The United States emphasizing the Human Development Index of |New York citizens as well as the high risk of infection by children playing in public places.

## Conclusion

I would be glad to verify the conclusion after the revised version of the manuscript, considering the recommendations.

Minor comments

In our opinion, the epg count is less relevant than the frequency of infected cats. A unique T. cati for example, may shed a high number of eggs/day (more than 100.000). Then, presence of eggs may not mirror the infection burden of parasites. Nevertheless, the number of infected cats is a surprisingly and undoubtedly an “extraordinary” result in a developed country.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2026 Jul 8;21(7):e0351437. doi: 10.1371/journal.pone.0351437.r002

Author response to Decision Letter 1

12 May 2026

Response to the Academic Editor / Journal Requirements

1. PLOS ONE style requirements and file naming.

The revised manuscript and front matter have been reformatted to follow the PLOS ONE main-body and title-page templates. Files have been renamed per submission guidance: Manuscript.docx, Revised Manuscript with Track Changes.docx, Response to Reviewers.docx, Cover Letter.docx, and Fig1.tif.

2, 4, 5. Funding statement, Role of Funder, and author–funder relationship. The funding sentence has been removed from the Acknowledgments. We request that the online funding statement be amended to:

“This work was supported by institutional startup funds awarded to PK from Long Island University, College of Veterinary Medicine, and from Rowan University, Shreiber School of Veterinary Medicine. The funders provided support in the form of salary for author PK but had no additional role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of all authors are articulated in the ‘author contributions’ section.”

We confirm in this cover letter that we agree with the standard PLOS funder statement and that the author contributions in the online form reflect each author’s specific role.

3. Mismatch between Funding Information and Financial Disclosure. There were no external grant numbers; both awards are unnumbered institutional startup funds. The placeholder grant numbers have been removed from the Funding Information section so that it now matches the Financial Disclosure verbatim.

6. Data Availability. We have prepared a complete minimal dataset comprising (i) the de-identified individual-cat dataset used in all analyses, (ii) the R analysis script that reproduces the results. (iv) a data dictionary, and (v) a dual licence (CC-BY 4.0 for data, MIT for code). These files have been deposited at Zenodo (DOI: [https://doi.org/10.5281/zenodo.19866813 ]).

The Data Availability Statement now reads:

“All de-identified data and analysis code required to reproduce the results are publicly available at Zenodo (https://doi.org/10.5281/zenodo.19866812). Trap-site coordinates have been rounded to two decimal places in the public dataset to protect free-roaming cat colonies; full-precision coordinates are available from the corresponding author on reasonable request, subject to a data-use agreement.”

7. Figure 1 copyright (map images). We agree this is an important compliance issue. The original Figure 1 base layer was generated in Google Earth/Google Maps and is not redistributable under CC-BY 4.0. We have replaced Figure 1 entirely with a new map prepared in QGIS 3.34 using only spatial data and basemap tiles whose licences are compatible with PLOS ONE’s CC-BY 4.0 publication terms:

• New York City borough/county boundaries — New York State GIS Clearinghouse (Counties_Shoreline.shp), public domain.

• U.S. Census Bureau TIGER/Line 2022 state boundary shapefile, public domain.

• Sampling points — our own field-collected coordinates, this study (CC-BY 4.0).

• OpenStreetMap Standard XYZ tiles for street/landmark context (© OpenStreetMap contributors, ODbL 1.0).

• CARTO Positron XYZ tiles for the muted reference basemap (© CARTO, CC-BY 4.0; underlying data © OpenStreetMap contributors).

Both basemap providers permit use in scholarly publications under their open licences provided the attribution is given in the figure caption, which we have done. The figure caption has been rewritten accordingly. The replacement TIFF meets PLOS ONE’s technical specifications.

8. Citation of reviewer-recommended works. Where reviewers recommended specific citations, we evaluated each for relevance and incorporated those that strengthened the manuscript (see point-by-point responses below).

Response to Reviewer #1

We thank Reviewer #1 for the careful, expert reading and for the specific terminology corrections that have noticeably tightened the manuscript.

Comment 1.1 — EPG terminology. “Unfortunately, the reference to EPG measurement is not appropriate, as no quantitative method (e.g., McMaster) was applied. I understand that an estimation of the relative parasite burden was performed by counting parasite eggs across the entire preparation; however, this is not sufficient to be expressed using the strictly quantitative EPG metric. Therefore, I would recommend removing the EPG measure throughout the manuscript and adopting an ad hoc metric, such as ‘number of eggs per preparation.’”

We thank the reviewer for raising this methodological point. We respectfully wish to retain the EPG metric, because a clarification of our protocol resolves the concern. The original Methods text was insufficiently detailed and led to an understandable interpretation that only a simple single-step qualitative centrifugal flotation was performed. In fact, fecal samples were processed by the Wisconsin double-centrifugation flotation technique, in which a weighed fecal sample (~ 2 g) was mixed with water, strained, centrifuged to pellet the eggs, resuspended in Sheather’s sucrose flotation solution, and centrifuged a second time with a coverslip in place. The Wisconsin double-centrifugation flotation is a concentration method that is widely accepted in veterinary parasitology as a quantitative technique. Because the centrifugation step concentrates and recovers all helminth eggs and protozoal oocysts onto the coverslip, and because we systematically examined the entire 22 × 22 mm coverslip area, every egg recovered by the procedure was enumerated. Dividing this total count by the weight of the fecal sample used (recorded to 0.01 g for every sample) therefore yields a quantitative eggs-per-gram (EPG) value.

To address the reviewer’s concern, we have:

• Rewritten the Methods (Laboratory analyses) to make the Wisconsin double-centrifugation procedure and weighed-aliquot quantitation explicit, including the formula (EPG = total egg count on coverslip / weight of fecal aliquot in grams) and the relevant references [10, 30, 31].

• Clarified that the Wisconsin double-centrifugation flotation is a concentration method that is considered quantitative, in which the centrifugation step recovers all eggs onto the coverslip and counting the entire coverslip therefore provides a quantitative egg count.

• Retained “EPG” throughout as the unit, since the metric is quantitatively justified.

• Added an explicit Limitations sentence acknowledging that even quantitative coproscopic egg counts are imperfect proxies for adult-worm burden, given variation in per-female fecundity and intermittent shedding (this also addresses Reviewer #2’s parallel concern, see 2.6).

Comment 1.2 — Title should reference New York City. “I would suggest adding the study area to the title, namely, New York City.”

Accepted. Since the samples were mostly from the NYC boroughs, the title has been revised to:

“Zoonotic endoparasites and Toxoplasma gondii seropositivity in free-roaming cats (Felis catus) from the New York City boroughs”

Comment 1.3 — Misuse of “Occult Heartworm ELISA test.” “Please change ‘Occult Heartworm ELISA test was performed…’ to ‘An ELISA test was performed…’. Note that ‘occult’ refers to a heartworm infection in which microfilariae are not detected in the blood (not investigated in the present study).”

Accepted. The reviewer is correct; “occult” is a clinical descriptor of amicrofilaraemic infection, not an assay name. The sentence has been revised to:

“A commercial heartworm antigen ELISA was performed by the Cornell University Animal Health Diagnostic Center as part of their standard diagnostic service to detect circulating Dirofilaria immitis adult-female antigen.”

We note that the precise commercial kit and reagent details are proprietary to the Cornell AHDC service laboratory and are not publicly disclosed by them; we have therefore cited the AHDC as the assay source rather than naming a kit we cannot independently verify. This wording also follows common practice in veterinary epidemiology papers using AHDC-tested specimens.

Comment 1.4 — “Variant” → “species.” “Line 216. Please change ‘variant’ to ‘species.’”

Accepted; “canine variant T. canis” has been changed to “canine species T. canis.”

Comment 1.5 — Heartworm wording on line 294. “Please change ‘While our results suggest that patent adult heartworm infection is not currently hyper-endemic in NYC feral cats’ to ‘While our results suggest that heartworm infection is not currently endemic in NYC feral cats.’”

Accepted, with a small qualification we hope the reviewer will agree with: because antigen ELISA detects only patent female adult infection, we have written:

“While our results suggest that heartworm infection is not currently endemic in NYC free-roaming cats, the antigen-only screening approach used here cannot rule out subclinical, single-sex, or pre-patent infection.”

This both adopts the reviewer’s recommended phrasing and preserves diagnostic accuracy.

Comment 1.6 — Knott’s test limitation in the Discussion. “Please add to the Discussion the limitation of not performing the Knott’s test… laboratory diagnosis in cats should include all possible tests, i.e. the Knott test, Ag detection and Ab detection.”

We thank the reviewer for raising this point. We agree that the modified Knott’s test is an important component of the full feline heartworm diagnostic triad, and we have added an explicit limitation paragraph. However, we respectfully note that the omission of the Knott’s test in this study was a deliberate methodological choice driven by sample-volume constraints, and we have explained the rationale in the revised Discussion. Specifically, three considerations drove the decision:

1. Limited blood volume. Free-roaming cats trapped under TNR conditions typically yield only small volumes of blood; the available volume had to be allocated across MAT serology for T. gondii, antigen ELISA for D. immitis, and qPCR for C. felis, leaving insufficient volume for the 1 mL of whole blood typically required for the modified Knott’s concentration step.

2. Cats are usually amicrofilaraemic. Feline heartworm infection is overwhelmingly amicrofilaraemic; published reviews report that naturally infected cats rarely produce detectable circulating microfilariae owing to low and often single-sex worm burdens and active immune-mediated microfilaricidal activity [29]. Antigen detection is therefore the recommended primary screening test in cats, particularly when sample volume is limiting.

3. Diagnostic prioritization under field constraints. Given (1) and (2), we prioritized antigen ELISA as the screening tool most likely to detect a patent adult infection in this small-volume cohort.

We have added the following paragraph to the heartworm subsection of the Discussion:

“A further limitation is that we did not perform the modified Knott’s concentration test or an antibody (Ab) ELISA. The Knott’s test was not performed because the small blood volumes obtainable from trapped free-roaming cats had to be prioritized across MAT serology for T. gondii, heartworm antigen testing, and qPCR for Cytauxzoon felis. In addition, feline heartworm infection is overwhelmingly amicrofilaraemic; naturally infected cats rarely produce detectable circulating microfilariae, owing to low and often single-sex worm burdens and active immune-mediated microfilaricidal activity [26], so antigen ELISA is the recommended primary screen in this host. Nevertheless, best practice combines antigen detection, antibody detection, and microscopic blood examination, and we recommend that future surveillance work in the region incorporate the full diagnostic triad whenever blood volume permits..”

We hope this addresses the reviewer’s concern by both acknowledging the limitation and providing a transparent biological rationale for the approach taken.

Response to Reviewer #2

We thank Reviewer #2 for the positive evaluation and for highlighting the One-Health and public-health framing of the work. The major and minor recommendations have all been incorporated.

Comment 2.1 — Materials and Methods: DNA extraction kit details. “kits used for extracting genetic material from faeces.”

We thank the reviewer for this important point and agree that complete reagent disclosure is the ideal. We respectfully note, however, that all molecular and antigen-based assays in this study were performed as fee-for-service tests by the Cornell University Animal Health Diagnostic Center (AHDC), a CLIA-certified and AAVLD-accredited veterinary diagnostic laboratory. AHDC uses validated in-house extraction and PCR protocols whose precise reagent components and primer sequences are proprietary to the laboratory and are not publicly disclosed; we therefore cannot provide the exact kit identifiers or primer sequences ourselves. We have, however:

• Revised the Methods (Antigen-based, serologic and molecular testing) to credit AHDC as the assay source, to make explicit that AHDC’s validated in-house protocols were used for DNA extraction and PCR, and to clarify which fecal, serum, and whole-blood subsets were submitted.

The relevant Methods text now reads:

“Antigen-positive samples were further submitted to the Cornell AHDC for molecular confirmation using their validated in-house PCR assays.

Serum samples (n=45) were screened for T. gondii antibodies using the modified agglutination test (MAT), with titers ≥1:25 considered evidence of prior exposure, consistent with previous studies in cats and wildlife [12, 13]. A commercial heartworm antigen ELISA was performed by the Cornell AHDC as part of their standard diagnostic service to detect circulating Dirofilaria immitis adult-female antigen.

EDTA whole-blood samples (n=45) were submitted to the Cornell AHDC for detection of Cytauxzoon felis using their validated in-house real-time PCR diagnostic assay.

We hope this provides sufficient methodological transparency while accurately reflecting that the analyses were conducted in an accredited diagnostic-laboratory setting where complete kit- and primer-level disclosure is not within the authors’ control.

Comment 2.2 — Describe primers used. “Describe the primers adopted in the trials (even in a supplementary file).”

We thank the reviewer for this request. We respectfully consider that we cannot appropriately provide a primer table in this manuscript, because we did not select or run the primers ourselves. The molecular assays were performed as fee-for-service tests by the Cornell AHDC using their validated in-house assays whose primer sequences and any in-house modifications are proprietary to the laboratory and are not publicly disclosed. We hope the reviewer will agree that this is the most accurate and honest representation of the work performed.

Comment 2.3 — ELISA test protocol for heartworm. “Describe, briefly, the ELISA test protocol for detection of Occult.”

Addressed jointly with Reviewer #1’s comment 1.3. The misnomer “Occult Heartworm ELISA” has been corrected, and the assay is now described as a commercial heartworm antigen ELISA targeting circulating D. immitis adult-female antigen, performed by the Cornell AHDC as part of their standard diagnostic service.

Comment 2.4 — Add a paragraph on human toxocariasis in the United States and risk to children in public spaces. “I suggest the inclusion of a paragraph/sentence concerning human toxocariasis in The United States emphasizing the Human Development Index of New York citizens as well as the high risk of infection by children playing in public places.”

Accepted. We have added the following paragraph to the Discussion, immediately after the Toxocara environmental-contamination paragraph:

“The public-health implications of these findings are particularly salient in a context such as New York City. Despite NYC’s high Human Development Index and substantial healthcare infrastructure, seroprevalence estimates indicate that approximately 5% of the U.S. population carries antibodies to Toxocara, with markedly higher rates among children, non-Hispanic Black populations, and residents of low-income urban neighborhoods [5]. Toxocariasis is recognized by the U.S. Centers for Disease Control and Prevention as one of five Neglected Parasitic Infections of public-health concern. Children are at disproport

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Decision Letter 1

Balbir B Singh

28 May 2026

Zoonotic endoparasites and Toxoplasma gondii   seropositivity in free-roaming cats (Felis catus  ) from New York City Boroughs

PONE-D-26-00846R1

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PONE-D-26-00846R1

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