Toxascaris leonina infected domestic cat (Felis catus) in Egypt; PCR-based molecular characterization of nematode eggs: a potential hazards to human health

Abstract

This molecular-epidemiological study was conducted in several locations in Cairo and Giza Governorates in domestic cats (Felis catus) to detect the most common intestinal helminths in feces and molecularly characterize this nematode. So, three hundred domestic cats were admitted to different clinics around Cairo and Giza Governorates with severe diarrhea, even watery, between January 2023 and April 2023. The ages of the cats ranged from 1 to 2.5 years old. Blood, sera, and urine samples were collected for further investigation of the health condition of the animals. Toxascaris leonina was the major intestinal parasite found in cat stools, with a prevalence rate of 5% (15 cats). Toxascaris leonina (T. leonina) eggs had oval elliptical surfaces and thick cuticles. An embryo was located inside the smooth outer shell wall of the shell. The animals suffer from normocytic normochromic anemia with leukocytosis, relative lymphocytosis, and thrombocytopenia. The amplification of the ITS-rDNA region from the ascaridoid nematodes was successfully performed using NC5 and NC2 primers. The PCR product of the ITS-rDNA fragment was sequenced and yielded 860 bp. The accession number of the sequenced ITS-rDNA region was OQ735413, submitted to Gene Bank, and based on the blast analysis of NCBI, the current ascaridoid nematode proved to be genetically related to the family Ascarididae and identified as T. leonina.

Introduction

The majority of nematodes (roundworms), which are common parasites that live in the gastrointestinal tracts of people, domestic animals, and wild animals, can result in significant financial losses and public health issues (Bugg et al. 1999; Chen et al. 2012a, b).

Toxascaris leonina (Nematoda: Ascarididae) is a common nematode of different animal species, including dogs, cats, wolves, tigers, lions, and foxes, which release eggs into the environment through their feces; Overgaauw (1997); Hajipour (2019).

Toxascaris leonina is a typical parasitic roundworm that infects foxes, dogs, and cats. Rodents like mice or rats serve as their intermediate hosts. When an animal consumes an infected rat, infection develops in the animal’s final host. Both dogs and cats can contract T. leonina, but cats are much more likely to have it (Okulewicz et al. 2012; Fava et al. 2020).

They have ingested eggs hatch in the small intestine of the final host. The larvae pierce the mucosal lining of the small intestine. They develop in the intestinal lumen after growth and molting. Eggs laid by the adult female worm are discharged into the animal’s feces. Three to six days in the environment are required for the eggs to become infectious. Cats can contract the disease by eating the egg or rodents that have infected larvae (Klockiewicz et al. 2019).

Although T. leonina, T. canis, and T. cati have well-known life cycles, those of T. malaysiensis still need to be fully elucidated. While T. leonina is transferred orally directly, T. canis and T. cati have oral and trans-mammary transmission and transplacental transmission for T. canis (Despommier (2003); Pawar et al. 2012).

Most Ascarididae nematodes wander to the lung and trachea to mature into adult worms (Soulsby 1982). In addition to T. canis and T. cati being their exclusive final hosts, a dog or a cat, respectively, they can also infect dogs and cats. However, several technical barriers, such as a need for knowledge about the genes that make up T. leonina, prevent development in many of these challenges. Numerous organisms, including parasitic nematodes like Brugia malayi (Blaxter et al. 2002), Toxocara canis (Maizels et al. 2000), Strongyloides ratti (Thompson et al. 2005), and Trichinella spp., have been used to characterize the transcribed genes of a particular organism using expressed sequence tags (ESTs) analysis and the single-pass sequencing.

So, this study aims to study the parasite epidemiology, diagnostics, and genetics identification of this vital feline parasite, which may be transmitted to humans.

Materials and methods

Sample collection

Three hundred domestic cats were admitted to different clinics (Pet Paradise Clinic, Winky Vet Clinic, and Kalb w Otta Clinic) around Cairo and Giza Governorates with severe diarrhea, even watery, between January 2023 and April 2023. Age of the cats ranged from 1–2.5 years old. Whole blood with EDTA (Salem et al. 2022a, b); sera and urine samples were collected and delivered to LeptoVet Lab for further blood work and urine analysis to check the health condition of the animals. The Institutional of the Animal Care and Use Committee of the Faculty of Vet Medicine approved this study CU. IACUC with number: Vet Cu 03162023728.

Fecal analysis

Fecal samples were placed in sterile plastic cups and kept at 4 C. Each sample Toxocara spp. Eggs were isolated using the concentration floatation techniques, as previously explained by (Soulsby 1982; Pritchard and Kruse 1982). Egg size was determined and photographed using Olympus BX50, Japan (Attia and Salaeh 2020).

Blood examination

Three hundred blood samples were collected from the domestic cats. EDTA whole blood and sera were collected for hematology and biochemical analysis (Zaki et al. 2021; Attia et al. 2021, 2022).

Hematological and biochemical analysis of the infected cats

Hematological analysis was carried out using Vetscan HM5 Abaxis hematology analyzer, Zoetis, USA, on 300 whole blood samples to detect the various blood parameters (RBCs count, Hb; hematocrit, platelets count, and WBCs count) that may be changed during infection. As well as Creatinine, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) using (Fujifilm dri-chem NX500V, Japan) were measured.

Molecular characterization of the isolated eggs

The collected eggs were rinsed three times with double distilled water H₂O. The samples were carefully mixed with 270 µl of tissue lysis (TL) buffer and 30 µl of proteinase K (50 ng/ml), then incubated at 55 °C for 15–18 h with shaking once every two hours. Following thorough digestion, DNA was extracted using the EZNA^@ tissue DNA extraction kit (Omega Bio-tek, Norcross, GA, USA), and the extracted DNA samples were kept at − 20 °C. The purified eggs were combined well with lysis buffer, and then 100 l of the mixture was added to several microcentrifuge tubes. The egg tubes were heated to 100 °C in a water bath for 5 min and then cooled to − 80 °C for 5 min. Five freezing and thawing cycles were followed by digestion with magnetic beads, and an EZNA tissue DNA extraction kit (Omega Bio-tek) was used to extract the eggs’ DNA. Before use, the samples were kept at − 20 °C (Chen et al. 2012a, b; Salem et al. 2022a, b). According to a prior study (Zhu et al. 2002), primers for T. leonina internal transcribed spacers (ITS) conserved sequences were created. ITS (NC5-forward: 5-GTAGGTGAACCTGCGGAAGGATCATT-3 and NC2-reverse: 5-TTAGTTTCTTTTCCTCCGCT-3); Chiu et al. 2021. 50 ng of template DNA, 2 ml of 2.5 mM dNTP (Takara Bio, Mountain View, CA, USA), 10 M forward and reverse primers (NC5 and NC2, respectively), 0.5 µl of rTaq DNA polymerase (also from Takara Bio), 5 µl of 10 Taq buffer, and 50 µl of ddH₂O were used to perform the PCR. The following were the PCR conditions: 95 °C for 5 min, then 35 cycles of 95 °C for 45 s, 55 °C for 1 min, and 72 °C for 1 min 30 s, with a final extension of 10 min at 72 °C in between (Attia and Salem 2022).

Results

Prevalence of the collected parasite

Ascaridoid eggs of T. leonina were the major intestinal parasites found in cats’ stools, with a prevalence rate of 5% (15 cats). T. leonina eggs had oval to elliptical surfaces and thick cuticles. An embryo was located inside the smooth outer shell wall of the shell. Due to the yolk membrane’s existence, the egg wall’s inside surface was rough or wavy (Fig. 1). The collected feces were diarrheic or even watery with severe mucous. The susceptible age was 13.33% in 1 year, 26.66% in 1.5 years, 26.66% in 2 years, and 33.33% in 2.5 years.

Fig. 1.

The eggs of T. leonina collected from cats faeces; the eggs measures 80 × 76 µm. which are oval; thick and smooth shell

The animals suffer from normocytic normochromic anemia with leukocytosis, relative lymphocytosis, and thrombocytopenia (Table 1).

Table 1.

Erthrogram levels of the infected cats with T. leonina

Parameters	Results		References range
R.B.Cs	5.02		5.0–10.0 million/c.mm
Haemoglobin	8.7		9.8–17.0 g/dl
Platelets	120.000		125.000–680.000/c mm
Haematocrit	26		30–45%
W.B.Cs	20,400		3500–20700/c mm
Differential Leucocytes count	Relative	Reference range	Absolute	Reference range
Basophils	0	0–1%	0	0–2 g/l
Eosinophils	2	2–10%	0.4	0.02–0.49 g/l
Staff	2
Segmented	50	45–64%	10.2	1.63–13.37 g/l
Lymphocytes	41	27–36%	8.3	0.83–9.1 g/l
Monocytes	5	0–5%	1.0	0.09–1.21 g/l
MCV	51.7		39–55 ft
MCH	16.3		13–17 pg
MCHC	33.4		30–36 g %
RDWc	15.5		14–18%

The kidney function tests were of normal range, but the liver function was elevated than the references range (Table 2).

Table 2.

Liver and Kidney function tests of the infected cats with T. leonina

Parameters	Results	References range
GPT (ALT)	380	22–84 U/L
GOT (AST)	104	18–51 U/L
BUN	24–30	19–34 mg/dl
Creatinine	0.9	0.8–2.2 mg/dl

The amplification of the ITS-rDNA region from the ascaridoid nematodes was successfully performed using NC5 and NC2 primers. The PCR product of the ITS-rDNA fragment was sequenced and yielded 860 bp. The accession number of the sequenced ITS-rDNA region was (OQ735413) issued by GenBank, and based on the blast analysis of NCBI, the current ascaridoid nematode proved to be genetically related to the family Ascarididae and identified as T. leonina. The BLAST alignment reveals that this ITS sequence displayed high nucleotide identity with T. leonina (99.65–95.97%; JF837178, MK309922, MK309897, MK309915O, M876356).

The phylogenetic tree was constructed based on the comparative alignments of ITS-rDNA of the current T. leonine against 22 different sequences of ITS-rDNA region belonging to Baylisascaris schroederi, Bayliss caristransfuga, Ascaris ovis, Ascaris lumbricoides, Ascaris suum, Parascaris equorum, Parascaris univalent, Toxascaris leonine, Toxocara canis, Toxocara cati, and Toxocara vitulorum. The phylogenetic relationship of these ITS sequences inferred two significant clades (Fig. 2). The first lineage was subdivided into two main branches. Our sequence of T. leonine was grouped with other sequences of T. leonina to form a monophyletic branch with a 98% bootstrap value and separated from other members of the family Ascorididae.

Fig. 2.

Phylogenetic tree constructed from the comparative analysis of ITS region sequences of T. leonine using the Neighbor-Joining model. N. americanus was used as an outgroup

Discussion

The most prevalent zoonotic gastrointestinal helminths infecting predatory mammals from the Canidae and Felidae families are Ascaridida nematodes, including T.canis, T.cati, T. malaysiensis, and T. leonina. An infected dog or cat excretes a large number of Toxocara spp. Eggs into the environment. As permanent hosts, these parasites infect mammals other than humans and rats. Infected eggs, earthworms, cockroaches, birds, and rodents with larvae in their tissues are the primary sources of infection. Typical human infection occurs when contaminated soil accidentally contains embryonated eggs. Visceral larva migrans, ocular larva migrans, hidden toxocariasis, and neurological toxocariasis are the four categories used to describe the clinical symptoms of toxocariasis (Lee et al. 2010).

Recent estimates for T. canis and T. cati infections in dogs and cats are consistent with the high prevalence of T. leonina infection estimated for the Eastern Mediterranean and African regions and low prevalence for the European, North American, and Western Pacific regions (Rostami et al. 2020).

Our study collected a low infection rate in domestic cats (5%), as they are indoors and at risk of eating rodents. In Iran, T. leonina infection rates were higher in stray dogs (6.6 vs. 1.5%) and cats (8.0 vs. 1.6%) compared to pet levels, suggesting that strays play a more significant role in contaminating the environment and spreading the disease. Even though T. leonina belongs to the same nematode family (Ascarididae), the greater prevalence in stray animals needed to be independently verified based on prior investigations of Toxocara species. Such animals are probable “persistent” reservoirs of T. leonina because they frequently have poor nutritional status, are prone to infections, are not cared for by a veterinarian, and are not given antiparasitic medications (Rostami et al. 2020).

Egg size was the morphological test employed in earlier studies to distinguish between different Toxocara spp.. However, this test is the least accurate way to distinguish between T. cati and T. canis. Many studies used PCR-based methods based on the ITS1 and ITS2 segments of rDNA to distinguish Toxocara spp. Eggs from adult helminths are closely related and physically identical (Khademvatan et al. 2013; Xie et al. 2020; Li et al. 2021).

Toxocara spp. Eggs were found in fecal and soil samples, using real-time PCR (2qPCR) technique to identify the species of the eggs. This method targeted the ITS2 sequences from adult worms. They asserted that soil and fecal samples might be used to detect the eggs of T. canis and T. cati using the recently established 2qPCR technique (Li et al. 2007; Paquet-Durand et al. 2007).

Toxocara spp. Eggs obtained from stray cats were identified using PCR with ITS2 primers; the findings were validated by sequencing. DNA analysis supports the findings of the current study.

In conclusion, Toxocara spp. is regarded as a neglected zoonosis, which frequently manifests in regions with low standards of cleanliness and education. This study attempted to educate veterinary and medical professionals about the need for intervention programs to lessen the burden of T. leonina and other ascaridoid infections in dogs and cats, mainly strays, focusing on reducing their transmission to paratenic or accidental host animals. It estimated the overall prevalence of T. leonina infection at 5% in cats. We can conduct some prevention and control measures in light of the intestinal parasites afflicting cats and dogs. The primary sources of infection for T. leonina are parasite eggs in the soil and feces. As a result, it is essential to clean up feces and sanitize the surrounding area regularly. Additionally, it is essential to consistently administer ivermectin, fenbendazole, and albendazole to affected tigers.

Authors Contribution

MA: The PI, Parasitology work, Molecular characterization, Editing, Idea. TM: Hematology work. OS: Chemistry work, Lab management. AA: Extraction and primers preparation, Lab management. AS: Editing, Manuscript Revision, Lab management.

Funding

No funding supporting this work.

Data Availability

All data are available in this manuscript. All the authors declare that all the data supporting the results reported in our article were found included in this article only.

Declarations

Conflict of interest

All authors declare no conflict of interest.

Ethical approval and consent to participate

This study was approved and follow the guide of the Ethical committee of the Cairo University; Faculty of Veterinary Medicine with number: Vet Cu 03162023728; this experiments were performed in compliance with the ARRIVE guidelines.

Consent to publish

Not Applicable.

Human and animal rights

I declare that the collection of samples from animals were conducted in accordance with local Ethical Committee laws and regulations as regards care and use of laboratory animals.