To My Quyen, Nguyen Khanh Thuan, Nguyen Phuc Khanh, Nguyen Thanh Lam*

1. Introduction

Isosporosis is a disease caused by a protozoan of the genus Isospora, which is responsible for causing diarrhea in dogs of several age groups and mainly in puppies (Santos et al., 2013; Schär et al., 2014). Infections of clinical importance are associated with the spreading capacity of Isospora spp. oocysts, observed in neonates or young animals; the main transmission route is fecal-oral (Joachim et al., 2018).

Mortality is approximately 30% to 40% in neonates. Among the causes of death are illnesses that involve dehydration, diarrhea, inadequate nutrition of the mother, and lack of hygienic-sanitary measures at the breeding place. The same factors are involved in the pathogenesis of isosporosis in dogs. However, different pathogenic agents, such as viral, bacterial, and parasitic (helminths and protozoa), are associated with acute canine gastroenteritis in puppies (Duijvestijn et al., 2016). Thus, diarrhea in neonatal dogs and puppies comprises several causes that should be better investigated with differential diagnostic methods, including important tools, such as coproparasitological techniques (Megid et al., 2016).

2. Aetiology

2.1 Morphology

Isospora species are coccidian protozoans that multiply sexually in the small intestine of the dog and secrete into feces oval oocysts lacking a micropyle, which is a small opening and a lid-like structure in the surface of the oocyst. The size of Isospora canis oocysts is 37×30μm, while Isospora ohioensis-complex oocysts are smaller, about 25×20μm. The species of I. ohioensis-complex parasites cannot be identified morphologically. In fresh feces, the oocysts are in unsporulated and in noninfective form, but they, after 1–4 days, become infectious through sporulation. A sporulated Isospora-type oocyst contains two sporocysts with four sporozoites inside each sporocyst (Figure 1) (Saari et al., 2018).

2.2 Classification

Isospora spp are protozoan parasites in the coccidian group that have been recognized as potential pathogens in dogs and cats for years (Dubey, 2009). The sexual phase of reproduction occurs in the gastrointestinal tract of dogs and cats, which culminates in the passage of unsporulated oocysts in feces. Dogs are the definitive hosts for I. canis, I. ohioensis, I. neorivolta, and I. burrowsi, and cats are the definitive hosts for I. felis and I. rivolta. The oocysts vary in microscopic appearance, which can be used to determine which species is causing the current infection (Table 1) (Lappin, 2010). 

Table 1: Isospora spp Oocyst Characteristics in dogs (Lappin, 2010)

3. Epidemiology

3.1 Geographic distribution

Dogs may be infected by Isospora or Cystoisospora canis (most frequently diagnosed), or by the Cystoisospora ohioensis complex, which is composed of morphologically undifferentiated structures, C. ohioensis and Cystoisopora burrowsi (Joachim et al., 2018). Although both may cause clinical disease, C. canis is the most pathogenic species, while the C. ohioensis complex is the most commonly detected. 

Cystoisospora species occur in dogs and cats around the world, including Canada. There are many species infecting these hosts, but in North America, the clinically most important are C. canis, C. ohioensis and C. burrowsi in dogs, and C. felis and C. rivolta in cats. Cystoisospora spp. were one of the most common endoparasites detected in a recent national survey of dogs and cats in shelters in Canada (Villeneuve et al., 2015). Oocysts were detected 10% - 16% in several states. Prevalence was higher in dogs and cats less than 1 year of age (dos Santos Reginaldo et al., 2019).

3.2 Transmissions

It is important to note that the main form of transmission is the fecal-oral route–the ingestion of non-sporulated oocysts eliminated with feces, which then contaminate the environment and food (MEGID et al., 2016). Puppies are definitive hosts that also become infected by the ingestion of tissue cysts present in paratenic hosts, such as rats and mice. Stressed or immunocompromised animals are more susceptible to this infection, as well as those animals that live in an overcrowded environment with poor sanitation (Megid et al., 2016).

Neonates become infected within the first three weeks of life, i.e., before weaning. However, at this stage of neonatal development, puppies are not able to eliminate feces by themselves and depend on the maternal stimulus of licking the perianal region. In most cases, the mother ingests the fecal contents, making it difficult to obtain these stool samples (Joachim et al., 2018).

4. Life cycle

Puppies are usually infected by ingesting sporulated Isospora oocysts from their living environment. If sporulated oocysts end up orally in dogs’ food, the freed sporozoites remain infectious and the dog may be infected by eating this paratenic host. The prepatent period of I. canis is 8–12 days and that of I. ohioensis complex parasites 4–9 days. The shell of the oocyst is broken down and the sporozoites are released in the intestinal tract. The sporozoites invade the epithelial cells of the small intestine as a kick-off of the asexual reproduction merogonia (also known as schizogonia), which takes place in several repeated cycles. Asexual cycles are followed by the phase of sexual reproduction: gametogonia. The end-result of the cycle is an oocyst, which is voided in the feces. These oocysts may number up to 200,000 in a gram of feces. In the environment, the oocysts sporulate in a few days, depending on conditions (e.g., in +20°C this takes 48 h) and subsequently become infective for their next hosts. The oocysts are viable and remain infectious for months. The lifecycle of Isospora spp. is illustrated in Figure 2 (Saari et al., 2018). 

(1) the dog is infected when sporulated oocysts of Isospora enter its gut; (2) the shell of the oocyst breaks down and releases parasitic sporozoites, which penetrate intestinal epithelial cells; (3) the asexual reproduction of the parasite (merogony/schizogony) begins in the epithelial cells of the small intestine, is repeated during several cycles, and leads to the infection of new epithelial cells; (4) the next state is sexual reproduction, or gametogonia, producing both male microgametes and female macrogametocytes; (5) the sexual cycle results in the fertilized zygote, which develops into an oocyst that exits the canine host in feces; and (6) the oocysts sporulate in the environment in a few days and become infectious to new dogs.

5. Pathogenesis

Coccidia show similar biological characteristics among themselves; however, four distinct endogenous stages are common to all species of Isospora – excystation, schizogony, gametogony, and sporogony. An extra-intestinal phase can also occur in definitive and paratenic hosts in which monozoic tissue cysts are formed, containing a sporozoite in the interior portion (called a hypnozoite) (Dubey et al., 2009).

Infection with Isospora spp. causes inflammation of the intestinal wall, with epithelial destruction. The transmission of this parasite occurs by the elimination of oocysts in the feces of infected animals that can contaminate the environment. In this manner, oocysts that are ingested with food or water settle in the gut and penetrate the epithelial cells. In combination with inadequate food, adverse climatic conditions, or poor conditions of management, a greater or lower severity of the disease can occur. While C. canis is located in the proximal portion of the small intestine, inducing enteritis due to direct lesion of the mucosa (due to schizogony and gametes), the C. ohioensis complex infects the enterocytes in the lamina propria of the small intestine, cecum, and colon, causing atrophy of intestinal villi, necrosis of enterocytes, and inflammation of intestinal crypts.

During the acute phase of infection, which occurs in the intestine, the occurrence of intense cellular necrosis can be observed. In the chronic phase, tissue cysts and mucosal atrophy are seen with inflammation of the lamina propria, loss of intestinal villi, and hyperplasia of Peyer’s plaques. After the complete endogenous development of Isospora spp., the oocysts are excreted through the feces and sporulate in the environment in a few days (Rauscher and Sch, 2013).

Immunity develops after the first infection and is more effective with advancing age of the exposed animals. Immunosuppression in dogs and cats facilitates reactivation of tissue cysts with release of sporozoites contained within these structures. Therefore, the enteric cycle restarts and causes recurrence of intestinal coccidiosis (dos Santos Reginaldo et al., 2019).

6. Clinical signs and pathology

6.1 Clinical signs

The clinical signs are due to destruction of the intestinal epithelial cells. The damage of the epithelium results in the diminution of the absorptive surface of the gut or full-thickness epithelial damage extending to the underlying connective tissue of the mucosa. This may be accompanied by hemorrhage into the lumen of the intestine and inflammation. The most important sign is watery or bloody, yet often self-limiting diarrhea, lasting sometimes for weeks. There may be vomiting and abdominal pain (Saari et al., 2018).

Clinical signs manifest typically in puppies under 4 months of age. The growth and development of the puppy may be affected even in subclinical cases. Coccidia are opportunistic pathogens; if pathogenic, their virulence may be influenced by various stressors such as changes in living conditions or feeding (e.g., when solid foods are introduced to puppies). Coinfections with viruses, bacteria, and other parasites are common in dogs displaying clinical signs (Saari et al., 2018).

6.2 Pathology

Cystoisospora canis and C. ohioensis sporozoites can be excysted in bile salts and trypsin like other coccidian parasites and used for in vitro studies. Limited multiplication occurred by endodyogeny in several cell lines; schizonts were not seen (Fayer and Mahrt, 1972)

Recent studies using C. canis sporozoites have demonstrated that monozoic tissue cysts would form in several cell lines of non-canine origin (Houk and Lindsay, 2013) (Figure 3). These monozoic cysts persisted up to 127 days, and zoites were motile after treatment with bile-trypsin excystation solution or acid-pepsin digestion solution (Figure 3) (Dubey and Lindsay, 2019).

For the oocyst-induced infections, eight dogs in a study were euthanized 6-120 h after feeding 1 million C. ohioensis sporocysts to each; to facilitate excystation of sporozoites in the gut of newborn pups, sporocysts had been liberated mechanically from sporulated oocysts. Irrespective of the inocula, stages were confined to surface epithelial enterocytes (Figure 4) First divisional stages occurred in the jejunum at 48 h post-inoculation (hpi). At 48 hpi, zoites occurred in pairs in parasitophorous vacuoles (pv) of surface epithelial cells of the jejunum. The pv were 7-9 x 6 µm and the zoites were 7-9 x 2-5 µm in sections. At 72 hpi, uninucleated zoites, multinucleated zoites and meronts containing fully formed merozoites occurred in surface epithelial cells of the jejunum. At 96, 114 and 120 hpi, asexual multiplication occurred throughout the small and large intestine, mostly in the ileum. The number of asexual generations was not determined. At least two structurally distinct meronts were identified at 96-120 hpi. Type I meronts contained larger merozoites (11 x 3 µm) than those (7-5 x 1-5 µm) in Type II meronts. Meronts were merozoite-shaped and contained up to 8 nuclei (Figure 5); multinucleated round meronts were not seen. Uninucleated, binucleated and multinucleated zoites occurred within the same pv. Gamonts occurred in surface epithelial cells of the small intestine, cecum and colon, but predominantly in the ileum, 96-120 h pi. Macrogamonts were 13-17 x 11-13 (14.5 x 12.8) µm in sections and 21-26 x 17-25 (21.7 x 17.6) µm in smears. Microgamonts were 13-17 x 8-15 (15.3 x 11.4) µm in sections and 24-30 x 15-24 (27 x 19) µm in smears and contained up to 50 microgametes (Figure 5).

The tissue cyst-induced cycle was also studied in six newborn dogs; the dogs were euthanized 6-120 h after feeding tissues of laboratory mice that had been infected with C. ohioensis oocysts 17 days earlier. Stages were not seen in the dogs killed at 6 and 12 h p.i. Asexual and sexual stages were structurally similar in size and location to the oocyst-induced cycle but their development was faster by 24 hpi in the mouse-induced cycle. Oocysts were excreted unsporulated (Figure 5). 

(A) Outpouring of the contents of lamina propria into the gut lumen. Arrow points to a meront; stages are not clearly visible at this magnification. (B) Higher magnification of an intact villus. Note, microgamont (arrow) and meronts (arrowheads).

7. Diagnosis

7.1 Morphological test

Cystoisospora infection in dogs can be diagnosed by identification of the unsporulated oocysts with any of the fecal flotation methods commonly used to diagnose parasitic infections. In dogs, only C. canis can be identified with certainty by oocyst size and shape. The oocysts of the other three other species of Cystoisospora namely C. ohioensis, C. burrowsi, and C. neorivota, may overlap in size, and their distinction is not clinically important. Rarely, epithelial casts may be found in feces; schizonts, merozoites, and partially formed oocysts can be found in smears made in normal saline (not water). Unsporulated oocysts measuring 10-14 µm should be considered Hammondia/Toxoplasma/Neospora and specific PCR assays are needed for definitive identification (Schares et al., 2005).

7.2 Polymerase chain reaction tests

Specific PCR assays are needed for definitive identification (Schares et al., 2005). Unlike other coccidia, Sarcocystis species oocysts/sporocysts are excreted sporulated. They would not be seen in dogs who were not fed meat or in puppies. Molecular data using the 18S rRNA and ITS 1 genes indicates close phylogenetic similarity between dog and cat Cystoisospora species and this information may be useful in differential diagnosis of C. ohioensis-like parasites (Lee et al., 2018).

8. Treatment

In diseases with acute gastrointestinal involvement, the first therapeutic measure is correction of water and electrolyte disturbances. Patients are presumed to have lost a sufficient amount of isotonic fluids until they become dehydrated due to diarrhea. Restoration of normal circulating volume is an immediate priority both to prevent functional renal damage, to minimize further damage to the gastrointestinal tract, as well as to maximize the action of drugs used for treatment. Thus, the administration of fluids in these patients is critical. 

Considering the pre-patent periods mentioned above, the definition of a treatment should consider two steps; the application of drugs that block the release of oocysts (metaphylaxis) and the improvement of clinical signs. The first step, which is the treatment administered during metaphylaxis, seems to be the most effective in the control of clinical signs (when there is still no lesion in the intestinal epithelium) and in the reduction of oocyst excretion. In order to evaluate a possible influence of other enteric pathogens of importance in puppies, especially Giardia spp., other diagnostic tests should be performed (Joachim et al., 2018).

The administration of sulfamethoxazole and trimethoprim, along with supportive therapies, such as restoring of physiological water and electrolyte balance, and the administration of metronidazole resulted in clinical recovery and control of spread of Isospora spp. oocysts (Neto and Kogia, 2015).

 However, sulfas are known to precipitate crystals in the renal tubules and promote acute interstitial nephritis by a sensitivity reaction, consequently leading to acute renal failure. Therefore, patients need rehydration, mainly for satisfactory drug action and correction of water imbalance. Table 1 lists the recommended drugs for canine therapy, regardless of the patient’s age. 

In conditions of acute diarrhea, the rates of water and electrolyte loss must be considered. In addition, it is necessary to measure serum and electrolyte replacement, especially potassium. The identification and correction of glycemic concentrations is always recommended in animals with clinical signs of weakness, hypothermia, and even stupor. This can be carried out by intermittent bolus or continuous infusion, associated with crystalloid solutions (Lee and Cohn, 2017).

9. Prophylaxis and control

Control measures involve the isolation of infected animals from animals with other types of diseases, reduction of overcrowding in kennels to avoid contact of healthy dogs with oocysts spread by infected dogs, and finally, awareness of an adequate hygienic environment and fomites with disinfection using solutions containing quaternary ammonia. Adequate disinfection of incubators, eaters, and drinkers is important. Moreover, personal hygiene measures between veterinarians and nurses need to be strictly followed, such as hand washing before and after handling the patient, as well as the use of gloves. If necessary (as in diarrheal cases), coats could be worn for further protection (Murtaugh et al., 2007). The control of insects where animals live also helps to reduce the number of infected animals, since insects can transmit oocysts from one environment to another (dos Santos Reginaldo et al., 2019).

10. References

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Duijvestijn, M., Mughini-Gras, L., Schuurman, N., Schijf, W., Wagenaar, J.A., Egberink, H., 2016. Enteropathogen infections in canine puppies: (Co-)occurrence, clinical relevance and risk factors. Veterinary microbiology 195, 115-122.

Fayer, R., Mahrt, J.L., 1972. Development of Isospora canis (Protozoa; Sporozoa) in cell culture. Zeitschrift für Parasitenkunde 38, 313-318.

Houk, A.E., Lindsay, D.S., 2013. Cystoisospora canis (Apicomplexa: Sarcocystidae): Development of monozoic tissue cysts in human cells, demonstration of egress of zoites from tissue cysts, and demonstration of repeat monozoic tissue cyst formation by zoites. Veterinary Parasitology 197, 455-461.

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