INTERNATIONAL JOURNAL OF VETERINARY AND ANIMAL MEDICINE (ISSN:2517-7362)

Cryptosporidium spp. is a parasite that has emerged all over the world as one of the main causes of diarrhea among animals. This protozoan is important not only because it encompasses a large number of hosts, but also because of its zoonotic potential. Cryptosporidium spp. oocysts which are eliminated in feces have infecting forms and high environmental resistance. They enable water, soil and food contamination while their ingestion leads to outbreaks that result in economic losses. These study aimed at evaluating the occurrence of Cryptosporidium spp. in calves in the south of Rio Grande do Sul state, Brazil. Three hundred fifty-nine samples of feces were directly collected at the rectal ampullae of calves that were up to one year old. Feces were processed by the Ritchie’s method, which was modified for sample sedimentation and staining by safranin methylene blue. Afterwards, they were analyzed by an optical microscope in the search for oocysts. Results showed that Cryptosporidium spp. was found in 6.69% (24) of samples. Twenty-four out of 68 farms under analysis exhibited Cryptosporidium spp. oocysts in animal feces, i. e., and 35.29%. Results led to the conclusion that infection caused by Cryptosporidium spp. occurs in bovine herds in different areas in the south of Brazil and that this species may act as an important source of infection of the zoonotic subtype of Cryptosporidium spp. to other species, mainly human beings.

Cryptosporidiosis; Bovines; Coccidia; Epidemiology; Zoonosis

Protozoa of the genus Cryptosporidium spp. are intestinal coccidia that inhabit a peculiar environment in their life cycle; their location may be intracellular, but extra-cytoplasmic [1].

Cryptosporidiosis is a globally distributed zoonosis caused by Cryptosporidium, a parasite that belongs to the phylum Apicomplexa, which causes enteritis and aqueous diarrhea [2]. Transmission results from consumption of water and contaminated food, besides inhalation of oocysts from the soil and self-infection [3].

Twenty-seven species of Cryptosporidium have been known so far [4,5]. The most common ones found in bovines are C. parvum, C. bovis, C. ryanae and C. andersoni [6-9]. The species C. parvum has been considered the agent that causes neonatal diarrhea in ruminants and human infection, a fact that characterizes it as zoonotic [10-12].

Bovine cryptosporidiosis was initially described in 1971 in animals whose clinical picture was chronic diarrhea [13]. In young bovines, infection caused by the protozoan may lead to growth retardation, mortality and consequent economic losses [14-16]. Calves of dairy cattle breeds which are up to two months old seem to be the main C. parvum reservoir; besides, practices of intensive farming may make transmission of this subtype easier [4,11].

This disease is one of the most common causes of diarrhea in calves which are up to thirty days old; it accounts for 37.2% of all cases [17]. The highest prevalence of infection occurs in animals which are up to two weeks old and it triggers neonatal diarrhea at variable intensities [18].

Transmission occurs through a fecal-oral route due to the ingestion of sporulated oocysts, as excreted by the host [19]. Cryptosporidiosis has been characterized by diarrhea, dehydration, poor absorption and weight loss [20]. According to Al-Braiken et al. [21], selflimiting infections may occur in immune competent individuals. In immune compromised individuals, the disease may become chronic and even cause death [22].

The size of Cryptosporidium spp. oocysts may range from 4 µm to 6 µm, a fact that hinders the diagnosis if a technique of auxiliary staining is not used [23]. Unlike other genera, such as Toxoplasma, Isospora, Eimeria and Sarcocystis, whose oocysts range between 10 µm and 40 µm, it is hard to identify the species based on size, shape and internal structures, since they are rather indistinguishable by an optical microscope [16,24,25]. Thus, staining fecal smear enables a more reliable, faster and simpler diagnosis to detect oocysts in fecal samples [26].

Some oocysts of species of Cryptosporidium spp., such as the ones of C. parvum, may keep viable for six months or more when they are in ideal conditions of humidity and temperature (20°C) [16]. In warmer environments (30°C), their viability decreases to three months, while they are destroyed at temperatures above 71.7°C. However, since they are resistant to cold, they may survive at negative temperatures, i. e., for several days when they are up to -10°C, and up to eight hours at -20°C [24]. Therefore, there are three important factors that contribute to infections and maintenance of the parasite: the large number of infective oocysts which are excreted to the environment, their environmental resistance and their high infectivity [4]. These factors, together with high animal density in current livestock production systems, enable cryptosporidiosis to take place, mainly when environmental conditions are favorable.

In the search for clarification of the importance of this coccidium in the environment under investigation, this study aimed at analysing the occurrence of infection caused by protozoa of the genus Cryptosporidium spp. in calves in the south of Rio Grande do Sul (RS) state, Brazil. 

Fecal samples were collected in 68 dairy farms located in 22 municipalities which are supervised by the Laboratório Regional de Diagnóstico (LRD), that belongs to the Veterinary School at the Universidade Federal de Pelotas, in Pelotas, RS, Brazil (Figure 1). Farms were randomly chosen in every municipality; at least 10 fecal samples were collected per farm and one fecal sample per animal.

Fecal samples of 359 calves, which were up to one year old, were collected directly from the rectal ampullae, stored, identified and sent to the Laboratório de Parasitologia Médica e Veterinária at the Universidade Federal de Pelotas. They were kept in MIF solution at temperatures that ranged from 2 to 8ºC. Sample homogenization was followed by liquefaction in sterile distilled water and smearing with 2 g feces per sample, in agreement with the technique described by Ritchie et al. [27] and modified by Young et al. [28]. Samples were fixed by a hot method and submitted to the method of Auramine-O Phenol, to be sorted out [29]. Afterwards, Ziehl-Neelsen staining was carried out to confirm positive samples [30].

Throughout sampling, people who oversee farms and/or management of animals were interviewed about the occurrence of parasitic enteritis and underdevelopment of young animals. Data were used for identifying relevant factors related to the epidemiology of cryptosporidiosis. This study was approved by the Animal Ethics Committee at the Universidade Federal de Pelotas, on June 6th, 2016 (no. 23110.0011818/2016-73).

Infection caused by Cryptosporidium spp. in bovines in Brazil was firstly studied in Botucatu, São Paulo (SP) state, Brazil. Oocysts were found in 26% of fecal samples collected from calves that were up to 30 days old, while 23% was found in calves that were over 30 days old [31].

Prevalence of Cryptosporidium spp. in bovines varies much; positive indexes in the literature range from 0.6 to 82.54%, but there are also reports of 100.0% [32,33]. In the study reported by this paper, occurrence of Cryptosporidium spp. in calves exhibited values below the ones found by studies carried out in Brazil, the USA, Hungary, Germany, Ireland, Iran and Spain, whose prevalence ranged between 14.0% and 57.8% [34-36].

Considering 359 fecal samples under analysis, Cryptosporidium spp. oocysts were identified in feces of 24 dairy calves (6.69%); 13 of them (3.62%) had diarrheic feces (two, i. e., 0.56%, was positive for oocysts), while 346 (96.38%) had non-diarrheic feces (22, i. e., 6.13% was positive for this protozoan in bovines). Regardless of the technique used for their detection, the incidence of oocysts was considered low by comparison with the ones found by other authors, such as Ederli et al. [37] and Watanabe et al. [38].

The study carried out by Ederli et al. [37] differs from those findings; they analyzed feces of 211 calves that were from four days to 12 months old, in a microregion named Campos dos Goytacazes, in Rio de Janeiro state, Brazil. Feces were examined by an optical microscope after concentration by centrifugation and staining by the modified Ziehl-Neelsen technique. Seventeen (8.06%) out of 211 calves under investigation had diarrhea; 2 (11.76%) were positive while 15 (88.24%) were negative for oocysts in their feces. Watanabe et al. [38] collected fecal samples of calves that were up to three months old and used the Ziehl-Neelsen staining technique to show that infection caused by Cryptosporidium in bovines was 37.6% (173/460). Ninety (46.40%) out of 194 calves – whose feces had normal consistency – exhibited oocysts in their feces.

Although elimination of oocysts was considered low in this study, by comparison with studies carried out in other Brazilian regions, the potential of this pathogen should not be underestimated. The infective form of this coccidium can be found in environments where bovines live and contamination sources are perpetuated not only because of the amount of feces expelled by these animals daily but also because oocysts survive in the environment for months, a fact that represents a source of infection, mainly in human beings [39].

Even though the technique of Auramine detected a larger number of positive samples than the one found by the Ziehl-Neelsen technique, it has a higher number of false positives, a fact that shows the need for more than one detection method, so as to mitigate the risk of false negatives and false positives. The use of the Auramine staining technique showed that 10.6% of samples had Cryptosporidium spp. oocysts, while the Ziehl-Neelsen one showed only 6.69%.

Twenty-four (35.29%) out of 68 farms, where fecal samples of calves were collected in order to search for Cryptosporidium spp. oocysts, had positive calves. This result corroborates the ones of other authors, who found that positivity of this pathogen ranged from 10 to 100% [40-41] and from 6.67 to 71.4% on farms under study [42].

On these farms, most animals are raised together, in calf sheds (54.04.3%; n=194), followed by the method of individual calf rearing, with the use of pens (37.05%; n=133). Both systems enable the environment to be highly contaminated with specific pathogens and their dissemination, as well.

This study showed that Cryptosporidium spp. oocysts were more frequent in young calves that were 168 days old (10.58%), on average, either kept in pens or raised on a pasture with several calves. Results were similar to the ones described by other authors, such as Maldonado-Camargo et al. [43], Faubert e Litvinski [44] and Silva Junior et al. [45]. It should be highlighted that most positive animals – regarding oocysts in fecal samples – did not exhibit any clinical signs of diarrhea and dehydration. Even so, the importance of these asymptomatic carriers may as disseminators of coccidia in the environment should be emphasized. Besides, asymptomatic carriers may undergo episodes of immunosuppression and stress, thus, triggering cryptosporidiosis.

According to Almeida et al. [46], collective water fountains used by animals also represent a significant risk factor that leads to Cryptosporidium spp. in calf feces. These structures of water supply may become reservoirs of oocysts that keep viable for a long time, besides not being affected by filtration and chlorination processes. On the dairy farms where this study was carried out, water fountains were shared between young and adult animals, except the ones that were in pens. As a result, they were an important factor in the contamination and dissemination of the disease among animals.

This study also showed that aggregation and overcrowding of calves in a certain place were determining factors that led to contamination in the environment and proliferation of microorganisms, since most animals were raised in collective calf sheds at their first months. According to Boufassa-Ouzrout et al. [47], transmission of cryptosporidiosis increases when there is either direct contact between susceptible animals and infected ones or indirect exposure of susceptible individuals to environments contaminated with Cryptosporidium spp. High calf density was also mentioned by Silva Júnior et al. [48] as a high risk factor of cryptosporidiosis because animals are exposed to a high load of parasites in the environment. Thus, it may be inferred that the place where animals are raised is an important source of contamination. Therefore, the use of good hygiene practices in calf rearing may decrease risk of infection caused by Cryptosporidium spp. and other pathogens which cause enteritis and affect young bovines [49,50].

According to Pohjola et al. [51], the most affected age range is the first week of life. Henriksen e Krough [52] and Bhat et al. [53] stated that cryptosporidiosis is more prevalent in calves that are from four to 30 days old. Such findings do not corroborate the ones of this study, which showed that older animals were proportionally more positive for oocysts in feces.

However, Mtambo et al. [54] conducted a study in which 5.3% of 486 fecal samples of bovines were positive for Cryptosporidium spp., a fact that corroborates data of the study reported by this paper, even though the occurrence of Cryptosporidium spp. oocysts was higher in calves that were under three months old, by comparison with weaned and adult ones. In this study, the highest occurrence was found in older animals, i. e., over 168 days old. These results may be explained by the fact that older animals were exposed to sources of infection for a longer period.

Absence of clinical manifestations observed in most animals whose feces had Cryptosporidium spp. oocysts, shown by several studies and data collection on coccidia, must be attributed to factors connected to the host, such as good nutritional status and absence of stressing factors, besides environmental contamination and pathogenicity of the different strains of this coccidium.

Concerning the sex of animals, this factor did not exert any influence on the level of infection caused by Cryptosporidium spp. in this study and corroborates findings of a study carried out by Tarazona et al. [55], who stated that the infection takes places regardless of sex and breed.

Twenty-four (6.99%) out of 359 fecal samples under analysis exhibited Cryptosporidium spp. oocysts. These findings were similar to the ones reported by Castro-Hermida et al. [56], who analyzed 379 samples of bovines that were from three to 14 years old and found 32 (8.4%) positive samples for Cryptosporidium spp. oocysts. This percentage was smaller than the ones found by Almeida [46], who reported 61%, and Venturini et al. [42], who observed 26.6%. Both studies evaluated animals that were up to one year old, i. e., the same age range that was investigated by this study, since it represents a category that is more prone to infection caused by Cryptosporidium spp.

Differences in results may be attributed to epidemiological variations, number of samples per farm, methodology of diagnosis and difficulty in recognizing oocysts. However, the most important factor is the intermittent pattern of oocyst excretion, a fact that may lead to data underestimation, mainly at the end of the infection. Therefore, all data must be taken into consideration to compare prevalence. The need for more than one staining method should also be highlighted in order to ensure high accuracy in the search for Cryptosporidium spp. oocysts in feces under examination.

Feitosa et al. [56] evaluated the prevalence of C. parvum oocysts in fecal samples of 459 calves that were up to 30 days old and in water and ground samples of calf sheds in 33 dairy farms in Araçatuba, SP, Brazil. The highest percentage of oocyst excretion was found in calves that were between eight and 14 days old (14.5%), while the lowest percentage (6.4%) was detected in the group of older animals (from 22 to 30 days old). In the study reported by this paper, results differ from the previously mentioned one, probably due to the age of the animals that ranged from five to 365 days and made most calves less favorable to infection caused by this protozoan, and because of the number of samples under analysis. Quadros et al. [57] analyzed fecal samples of calves that were up to three months old in Lages, Santa Catarina state, Brazil, and found positivity of 17% in 200 samples. These results were higher than the ones found by this study, probably because of the age range of the calves under investigation, since this category is considered to be more prone to infection caused by Cryptosporidium spp.

It should also be emphasized that climatic/environmental factors in the cited studies are different, thus, validating a distinct epidemiological reality from the area under the influence of the Laboratório Regional de Diagnóstico (LRD) in the south of RS, Brazil.

Concentration of dairy calves and environmental contamination are factors that contribute to the occurrence of cryptosporidiosis in their herds.

The high number of asymptomatic animals found in the farms under investigation enabled to show that farmers must be warned of cryptosporidiosis in order to make them use management practices that aim at mitigating environmental contamination so as to avoid economic losses during outbreaks.

Although low occurrence of the parasite was observed in fecal samples under analysis, the agent can be found in the dairy bovine herds in the south of RS, Brazil, a fact that may lead to infection of susceptible animals, such as neonatal calves, and risks to public health, since it has zoonotic potential.

The present work was carried out with the Coordination of Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001.

The authors would like to thank everyone who contributed to the study.

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