Cryptosporidiosis is a zoonotic protozoan disease that has a very broad and versatile geographic distribution including the Antarctic region . Cryptosporidium is the causative agent and infects mainly the intestinal tract and rarely the respiratory system of diverse species including human, ruminant, feline, canine, rodent, avian, reptile and fish. Transmission usually occurs through the direct fecal-oral route or through ingestion of water or food contaminated with oocysts [2,3].
Bovines are the most common species of mammals, infected with Cryptosporidium and considered the major reservoir of Cryptosporidium for human infections . Cryptosporidiosis in cattle is mainly caused by C. parvum, C. andersoni, C. bovis and C. ryanae [2,3]. At least 10 other Cryptosporidium species or genotypes such as C. felis, C. meleagridis and C. suis can also play role in etiology [4,5].
Bovine cryptosporidiosis is considered one of the most common causes of neonatal diarrhea in cattle, leading to economic losses. The severity of infection ranges from mild to severe depending on Cryptosporidium species as well as age, previous exposure and immune status of the host. Asymptomatic infection is common in yearling heifersand mature cows [6,7].
Cryptosporidium parvum is a zoonotic species and the predominant in preweaned calves, especially those at age of 1-4 weeks . The agent is responsible for about 85% of cryptosporidiosis in preweaned calves but only 1% of the disease in postweaned calves and 1-2 year old heifers [6,9,10]. Among the other bovine species, C. bovis and C. ryanae were detected mainly in weaned calves, and C. andersoni in yearlings and adult cattle [9,11,12]. While C. bovis and C. ryanae are considered non-zoonotic, C. andersoni has recently beenreported in few research involving humans in England .
The specific diagnosis of Cryptosporidium species is central to the control of the disease and to the understanding of the epidemiology. Lack of distinctive morphologic features of Cryptosporidium oocysts makes microscopical examination inconvenient in order to clearly differentiate species and genotypes . Traditionally, C. parvum has been diagnosed by microscopy of fecal smears, with or without staining. However, two other species, C. bovis and C. ryanae, with similar oocyst morphology to C. parvum, can only be identified using DNA analysis. That is, microscopy cannot distinguish these three species . Therefore, molecular analyses are required to detect and distinguish Cryptosporidium at species/genotype and subtype levels [3,14]. The most frequently used marker for Cryptosporidium species and genotype identification is the small subunit of ribosomal RNA (SSU-rRNA) gene .
The disease in calves has been studied in many countries, with prevalence ranging from 2.4 to 100% [3,7]. In Turkey, cryptosporidiosis was first diagnosed in calves in 1984 . Since then, other surveys have revealed the prevalence of 7.2-63.9% in calves [16,17]. Most of the studies carried out in Turkey were based on microscopy of stained oocysts in feces [15,16,18-21]. Enzyme-linked immunosorbent assay (ELISA) [17,22] and PCR technique [23-25] have recently become more common to attain the prevalence of cryptosporidiosis. However, few studies have coped with genetic structure of Cryptosporidium species in Turkey [26-28].This study was conducted to determine the prevalence of cryptosporidiosis and to characterize Cryptosporidium species based on PCR amplification and sequence analysis of SSU rRNA gene in younger than 1-month-old calves in Erzurum province, Turkey.
Material and methods
A total of 307 fecal samples were collected from calves less than 1 month old in dairy farms (herd size ranging from 100 to 350 Brown Swiss, and Holstein cows in 3 professional dairy farms and from 40 to 85 Brown Swiss, crossbreed, and Anatolian Red cows in 2 traditional dairy farms) located in Erzurum province between April-2010 and October-2010. Samples were collected directly from the rectum with a gloved hand and transferred into a plastic cup. Fecal consistency was scored as firm, well formed, loose and diarrheic. Samples were kept at 4°C until laboratory analyses.
The study protocol was approved by the Animal Care and Use Committee at Ataturk University (4.4.2008-2008/8 decision number).
DNA Extraction and PCR Amplification
Oocysts were washed and concentrated from feces [29,30]prior to DNA isolation using a QIAamp DNA Stool kit (Qiagen, Maryland, USA). Before eluting, aliquots were added with 100 ml Buffer AE and stored at 20°C.
A nested PCR for the amplification of a fragment of SSU rRNA gene was performed using the protocols and primers as described by Xiao et al. with the following modifications: At the first step of nested PCR, approximately1.325 bp PCR product was amplified using primers 5’-TTCTAGAGCTAATACATGCG-3’ and 5’-CCCATTTCCTTCGAAA CAGGA-3’. The PCR contained 1x PCR buffer, 6 mM MgCl2, 0.2 mM (each) dNTP, 200 nM (each) primer, 0.025 U of Taq DNA polymerase, and 1.5 μl of DNA template in a total 25 μl reaction mixture. A total of 35 cycles were carried out at 94°C for 45 s, 55°C for 45 s and 72°C for 1 min. There was also an initial hot start at 94°C for 3 min and a final extension at 72°C for 7 min. A secondary PCR was then performed to amplify 826-864 bp from 1 μl of the primary PCR mixture using primers 5’-GGAAGGGTTGTATTTATTAGATAAAG-3’ and5’-AAGGAGTAAGGAACAACCTCCA-3’. The PCR and cycling conditions were identical to the primary PCR. Amplification products were separated by electrophoresis on 1% (w/v) agarose gels, and visualized by ethidium bromide staining.
DNA Sequence Analysis and Phylogenetic Analysis
Successfully amplified samples were subjected to DNA sequence analysis for species determination. Sequencing was performed using the ABI PRISM® BigDye terminator cycle sequencing kit in ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA). Sequence data were then subjected to BLASTN (RefSeq) searches of the Cryptosporidium genome database at the National Center for Biotechnology Information (http://www.ncbi nlm.nih.gov/). All sequence data were edited using BioEdit 7.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and FinchTV Version 1.4.0 (http://www.geospiza.com/finchtv) following naked eye checking. Multiple sequence alignments were made with the Clustal W method with BioEdit 7.0 software . The neighbor-joining (NJ) method as implemented in the MEGA5.1 program  was used for the phylogenetic analysis based on SSU rRNA, utilizing Eimeria tenella sequence (HQ680474) as out-group. The branch reliability was assessed by the bootstrap method with 1000 replications.
The PROC MEANS and FREQ were computed to obtain descriptive statistics . Animals were categorized by breed (culture, crossbreed and local), age (1-7, 8-15 and 16-30 days) and fecal consistency (firm, well formed, loose and diarrheic) before establishing cross-tables to reveal association of risk factors with cryptosporidiosis using Chi-square. The associations were considered significant at P<0.05.
The nested PCR showed a total of 12 (3.9%) positive amplifications in 307 fecal samples (Table 1). The cryptosporidiosis prevalence among calves raised in modern farm was lower than those raised in farms with poor infrastructure (1.5 vs. 8.5%, P<0.003). The infection rate in culture breed was insignificantly higher (4.5%) than the other breeds.
As the age advanced, the frequency of animals infected by Cryptosporidium increased quadratically, being the highest among calves aged 8-15 days (P<0.0001). Cryptosporidiosis was more common in females than males (6.8 vs. 1.3%, P<0.01). The cryptosporidiosis prevalence increased with the fecal water content (P < 0.0001), 13.6% in calves with the diarrheic feces and 7.1% in calves with the loose feces (Table 1).
All PCR-positive isolates in Erzurum were confirmed to be C. parvum (Fig. 1). The sequences were deposited into GenBank under accession numbers KC437395 to KC437406, respectively. C. parvum [GenBank: JN245618, JQ250804, JX886767, DQ656355, AB513881, JX948126, JX416362, JQ413434, JQ182993], C. andersoni [GenBank: JX948125, JX437080], C. bovis [GenBank: JX515546, JX886773, JN245624] and C. ryanae [GenBank: JX886771, JN245623] were reference species, whereas E. tenella (HQ680474) was an out-group reference in comparisons. Fig. 1 depicts phylogenetic relationship among C. parvum in Erzurum isolates and the other Cryptosporidium isolates as inferred by the NJ analysis of the partial SSU rRNA gene sequences. C. parvum in Erzurum isolates were grouped into the same clade with respective reference C. parvum sequences. In the present experiment, the percent identities were 99.3-100% among C. parvum in Erzurum isolates, 98.7-100% with other C. parvum isolates and 87.8-94% with other Cryptosporidium species from GenBank.
Fig 1. Phylogenetic relationship among Cryptosporidium isolates as inferred by neighbor-joining analysis of SSU rRNA nucleotide sequences. The sequence for E. tenella (HQ680474) was used as an out-group. Numbers on branches indicate percent bootstrap values from 1000 replicates. All of Erzurum isolates were identified as C. parvum.
The prevalence of cryptosporidiosis in Turkey varies between 7.2-63.9% in calves [16,17]. To our knowledge, this study delivered the lowest prevalence rate (3.9%) among other reports from different locations of Turkey [17,19-22,24,26-28].The difference could be due to a vast number of factors such as breed, age, management, environment, and season as well as diagnostic method [5,7,13]. The low prevalence could also be caused by spot fecal sampling instead of serial sampling, which may result in underestimation because of intermittent oocyst excretion [9,11].
The majority of C. parvum infections appear to be limited to dairy calves under eight weeks of age [10,35], being highest in calves up to 1-month-old [7,8,36]. In calves, the highest infection rates are reported in calves 7-14 days old [7,37],8-14 days old [4,38] and 8-21 days old . In accordance with the literature, in the present study, the infection prevalence was highest in calves aged between 8-15 days (12.7%), followed by those aged 1-7 days (6.7%) and 16-30 days (0.5%).
As previously reported by Trotz-Williams et al. in Ontario, Canada, by Aysul et al. in Aydin, Turkey andby Coklin et al. in Prince Edward Island, Canada, C. parvum was the only species identified in calves less than 1 month old. On the other hand, the absence of C. bovis, C. andersoni and C. bovis in our study could be a result of the age group (≤ 1 months) because since C. bovis and C. ryanae are known to be more prevalent in weaned calves and C. andersoni in yearlings and adult cattle [6,9,11,12].
Calf diarrhea has a multifactorial etiology, and C. parvum is frequently associated with the disease [7,38,39,41]. Besides, viruses and bacteria are other causative agents that can cause this symptom simultaneously or individually. Of 12 C. parvum positive fecal samples, 8 were from diarrheic calves and 4 from calves with loose feces (Table 1). In disagreement with some previous studies [35,39,41,42], our results proved an association of fecal consistency with the infection. Studies reporting relationship between fecal consistency and cryptosporidiosis are available [7,12,36]. Because other possible agents were not searched in the present study, it requires caution to make inference that calves with watery feces are prone to cryptosporidiosis. Another factor to contribute fecal dry matter is feeding scheme because looser feces can be consequence of milk feeding . These suggest that extensive sample analysis is required to confirm the relationship between fecal consistency and cryptosporidiosis.
The molecular characterization of Cryptosporidium species in Turkey has been published in three reports, in which C. parvum [26-28], C. bovis  and C. ryanae  were identified. In our study, homology search proved that all isolates in Erzurum were C. parvum. The partial SSU rRNA gene sequences had 100% similarity to reference sequences downloaded from the GenBank (DQ656355, AB513881, JX948126, JX416362, JQ413434, JQ182993 and JN245618). The NJ phylogenetic analysis based on the SSU rRNA (Fig. 1) showed that all sequences of C. parvum in Erzurum isolates clustered in the intestinal clade with reference C. parvum sequences (bootstrap value 92).
In conclusion, the current study elucidated the prevalence of cryptosporidiosis and the molecular characterization of Cryptosporidium species found in calves in Erzurum, Turkey. The prevalence of Cryptosporidium infection in dairy calves determined by nested PCR was at 3.9%. C. parvum was the only causative Cryptosporidium species in calves younger than 1 month in Erzurum province as ascertained by sequencing the amplified SSU rRNA regions.
The authors thank Mehmet Özkan Timurkan for technical assistance.
This article was originally published in Kafkas Univ Vet Fak Derg 19 (6): 969-974, 2013 DOI: 10.9775/kvfd.2013.9187. Reproduced with permission from the author.
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