Prevalence of Cystoisospora spp. in Domestic Cats in Mexico

Prevalence Abstract Cystoisospora spp. is a parasite with high transmission potential. Among populations of the family Felidae , Cystoisospora felis and Cystoisospora rivolta are the species that affect the domestic cat. Cystoisosporosis is characterized by high morbidity and low mortality; immunocompromised patients experience more severe and prolonged signs. The prevalence of Cystoisospora varies according to the study region; prevalences from 1.4 to 21.9% have been reported in cats within households and up to 46.3% in stray cats. The objective of this study was to determine the prevalence of Cystoisospora in cats with owners from different states of Mexico. Fecal samples (n= 927) from cats attending consultation from June to December 2019 were collected. 429 were positive: 13.59% came from kittens (under 6 months old), 9.49% from juveniles (aged 7 to 12 months), and 27.51% from adults (over 13 months). There was a prevalence of 50.59%; risk factors included an outdoor lifestyle, having been adopted, and lack of grooming. These could be associated with coexistence with other cats having access to the outside, the consumption of infected tissues, or contact with contaminated areas, representing the main forms of transmission of this protozoan.


Introduction
The cat (Felis catus) is a domestic species that has been coexisting with humans for millennia [1]. It has maintained its hunting habits, mainly for rodents, reptiles, birds, fish, and amphibians; over the past two decades, ownership of cats as pets has increased, and so has the adoption of stray specimens., Thus, cats have gained a place in homes around the world, becoming part of the family nucleus [2,3].
However, they commonly have access to the outside world, which makes them susceptible to various enter oparasites, such as protozoans. Among these, the genus Cystoisospora represents a high transmission potential, mainly among populations of the family Felidae, since it is species-specific. Cystoisospora felis and Cystoisospora rivolta are the species affecting the domestic cat [4,5]; these coccidias are transmitted efficiently by ingestion of sporulated oocysts present in the environment or by ingestion of contaminated tissues. They multiply intracellularly in the enterocytes, producing different degrees of epithelial damage; but immunocompromised patients experience more severe and prolonged signs, making therapeutic management difficult. This is generally true for stray cats and animals in overcrowded conditions and/or with nutritional deficiencies, which can be reservoirs and sources of infection [9]. Prevalence's from 1.4 to 21.9% have been reported in cats within households [10]. The objective of this study was to determine the prevalence of Cystoisospora spp. in owned cats in Mexico.

Materials and Methods, Area Descriptions
A cross-sectional study was conducted in 18 of the 32 states of Mexico (Table 1) from June to December 2019. In total, 927 fecal samples of cats were collected for consultation, regardless of breed, age, gender, provenance, or health status; all owners who agreed to participate in the project were asked to sign an informed consent and to complete a survey about epidemiological data and risk factors for Cystoisospora.

Sample Collection
The samples were collected from 178 clinics, hospitals, and veterinary offices, with the help of 305 previously trained veterinary doctors (training was provided through workshops, videos, conferences, webinars, and communication via email or WhatsApp) All obtained data were stored in an Excel sheet, and the analyzed samples were verified through electronic photographs evaluated by the researcher.

Analysis Procedures, Cyst Recovery Techniques, and Coproparasitoscopic Analysis
The fecal samples were collected fresh and subsequently processed directly in the veterinary clinics, using direct examination as well as the Faust and Graham techniques. For direct examination, one drop of physiological saline was applied on a slide strip, and at the other end, one drop of Lugol´s solution and a small, una pequeña fresh fecal sample (1mg of feces) were placed and mixed homogeneously with each solution. The samples were cover with a cover slip and observed under the microscope [11,12] report a sensitivity of 97.1 % and a specificity of 93.8% of this technique for protozoans. For Graham's method, one sample was obtained with acetate or scotch tape from the anal region of the cat, adhered to the slide sheet, and subsequently observed under the microscope [13]. Beltran et al. 2020, report a sensitivity of 71% and a specificity of 100% for this simple, raid, and economic method; however, it cannot be performed in stressed and aggressive cats and are very inefficient [14]. Faust's technique was performed by suspending 1 to 1.5 g of feces in distilled water, followed by sieving and centrifugation at 1,500-2,000 rpm for 2 minutes. The supernatant was discarded, and 2 to 3 ml of 33% zinc sulfate solution (specific gravity-1.18) were added; the mixture was stirred until the sediment was completely suspended. It was then centrifuged again at 2,000 rpm for 2 minutes. Using a bacteriological handle, a sample was taken from the tube and placed on a cover slip with a drop of Lugol´s solution [15] and a few drops of zinc sulfate; the tube was left in the centrifuge until a meniscus was achieved and subsequently covered for a few minutes. After this, one drop of Lugol´s solution was applied to a slide, covered with a cover slip, and observed under the microscope. Previous studies have reported a sensitivity of 100% and a specificity of 83% [16] for this method. It is inexpensive and keeps the sample clean, making it more reliable.
However, the procedure is more time-consuming, and there may be greater chances of errors during processing, such as dilution of the zinc sulfate solution and damaging of cysts after prolonged contact with the solution. Inadequate management of the final centrifugal solution makes it difficult to identify species; thus, the possibility of observing co-infections is diminished.

Statistical Analysis
The results for each sample were recorded in an Excel Microsoft Office sheet. The prevalence of isolated parasites was estimated as the number of positive animals divided by the total number of animals examined.

Results
Age was not associated with prevalence, but it was a risk factor (OR 1.47, P. 0.01) for kittens (Table 1). Coexistence with other cats translated into a tendency to be positive to Cystoisospora, but there was no association, nor was it a risk factor. Living with other animals and having a hunting habit were not associated with prevalence nor were they a risk factor in these felines (Table 1) [18]. Our study revealed a prevalence of 50.59%, which is very high compared to the 1.4% reported by [11] in Japan, to the 5.8% in Canada [19], to the 9.4% in North America, to the 9.5% [20] and 16.4% [21] in Greece, and to the 219% in Germany [22].

Copy@ Ariadna Flores Ortega
It is estimated that stray, shelter, and outdoor cats are more susceptible to parasitic infections due to their predatory habit, as they consume rodents, birds, reptiles, amphibians, and even food scraps and are also in greater contact with other cats; they are also factor, since the hair may contain a significant proportion of parasite eggs, which is in agreement with [3]. Considering that most cats are adopted and that the majority of them come from the street, they may have discharged cysts from these protozoans into the environment. Previous studies have revealed that large numbers of infectious eggs from other parasites are detected in the fur [25], suggesting that direct contact with the fur of a contaminated cat could be an additional route of transmission and re-infection among the feline population [26].

Conclusion
The data presented in this analysis show that Cystoisospora spp.
was present in most of the samples from owned cats. The prevalence rate was 50.59 %, which is high even compared to other studies conducted in stray cats. We also showed that cats with access to the outside or which have been adopted have a higher incidence of these protozoans; in addition, brushing the pet decreases the numbers of these parasites. Although they do not produce zoonoses, our data suggest the need to improve the control measures for these coccidian's to decrease their morbidity, including effective therapeutic management procedures, stress management, feline and environmental hygiene, in addition to paratenic host control.