First Molecular Genotyping of Cryptosporidium felis in Cattle, Iraq

1. Introduction

Cryptosporidium genus is one of the most important intracellular protozoan parasites, which belongs to the Eucoccidiorida Order of Apicomplexa Phylum. In the 1907s, the parasite was detected for the first time in mice ( 1 ), while in cattle and humans, it was first isolated in 1970 ( 2 ) and 1976 ( 3 ), respectively. Approximately 30 species of Cryptosporidium are recently recognized to infect mammals, fish, reptiles and birds. These species are classified according to host specificity, life cycle, morphology and the size of the oocysts ( 4 ). Additionally, the species of this parasite shown to have several unique characteristics that differentiate them from other protozoa, such as innate resistance to disinfectants, ability to initiate self-infection, and their ability to be unusually located in the host cell, cell membrane and sequestrations between cytoplasm ( 5 ).

In the last 20 years, although there have been dramatic increases in studies about cryptosporidiosis, critical questions concerning the epidemiology, life cycle, transmission, cell invasion, and host-parasite interaction remain unclear. This parasite appears to be transmitted due to contact with infected animals ( 6 ). Canine and felines are the usual hosts of Cryptosporidium and are thought to act as a reservoir ( 7 ). In 1979, C. felis was discovered firstly in a cat that acts as a major host ( 8 ), but humans and cattle can be infected as minor hosts ( 9 ). In cattle, newborns and calves showed a higher susceptibility to cryptosporidiosis shortly post birth and were still infected for weeks or months, as identified in dairy and beef cattle ( 10 ).

Early epidemiological reports were applied to detect the morphology and immunology of the parasite without sub-typing of Cryptosporidium spp.; but in the last decade, molecular diagnostic techniques have been used extensively as they are highly sensitive and specific in the identification of host species and genotyping ( 7 ). Polymerase chain reaction (PCR) assay was the almost molecular method employed for detecting Cryptosporidium oocyst and determining the species and genotype in many clinical and environmental samples ( 11 ). Sequencing of DNA considers a gold standard way for identification of mutation rapidly and for discovering polymorphism in a single nucleotide ( 12 ). To date, sequencing is used to enable Cryptosporidium classification, compare genetic materials and phenotypes differences, and evaluate genetic variations within Cryptosporidium spp. ( 13 ).

In Iraq, many studies were carried out to detect Cryptosporidium spp. ( 14 , 15 ), however, there were neither online data nor reports about C. felis in any animal or human. Hence, this study was designed to demonstrate C. felis infections in cattle using the molecular PCR assay and to document some local isolates in the NCBI for the first time in Iraq. The relationship of positive results to clinical and demographic risk factors of study animals was also aimed.

2. Materials and Methods

2.1. Samples and Data Collection

Overall, 130 female and male diarrheic calves of ≤1 year old were selected from different sub-urban and rural regions in Wasit province (Iraq) from February to April (2021). Each study animal was subjected to directly sampling fresh fecal (about 50 gm) into a labelled plastic container that was transferred cooled to the laboratory. Also, all study animals were examined clinically to estimate their body temperature, pulse and respiratory rates.

2.2. Molecular Assay

About 1 gm of each fecal sample was subjected to extraction of the DNAs following the manufacturer’s instructions for the Presto^TM Stool DNA Extraction Kit (Geneaid, Korea). The purity and concentration of extracted DNAs were checked using the Nanodrop system (Thermo-Scientific, UK).

Targeting the HSP70 gene of C. felis, one set of specific primers [(F: 5′-AACCTGGTTGATCC TGCCAGTAGTC-3′) and (R: 5′-CCCATTTCCTTCGAAACAGGA-3′)] was designed ( 16 ) and provided by the Scientific Researcher Co. Ltd. (Iraq). Then, the Mastermix tubes were prepared using a ready-to-use Go Taq Green Master Mix Kit (Promega, USA) at a final volume of 25 l, to be subjected later to the conditions of Thermal Cycler (Bio-Rad, USA) as follows: 1 cycle initial denaturation (95°C / 2 minutes), 33 cycles for denaturation (95°C / 30 seconds), annealing (60°C / 30 seconds) and extension (72°C / 50 seconds), and 1 cycle final extension (72°C / 5 minutes). The PCR products were analyzed using the stained 1.5% agarose gel with Ethidium bromide at 100 volts and 80 Am for 1 hour. The positive samples were amplified at 480bp.

Five positive PCR products were selected randomly for sequencing at the Macrogen Company (Korea) using the Sanger method, and the received data were submitted later for documentation in the NCBI. The local isolates were aligned with the NCBI-BLAST isolates for phylogenetic tree analysis and homologic identity.

2.3. Statistical Analysis

All data were analyzed using the GraphPad Prism (GraphPad Software Inc., USA) by One-way ANOVA test and t-test. Values of clinical examination were represented as meanstandard deviation (MSD). Statistically, significant differences were detected at P<0.05 ( 17 ).

3. Results

23 (17.69%) diarrheic calves were positive for C. felis targeting the HSP70 gene (Figure 1). Concerning clinical examination, positive calves to C. felis revealed insignificant variation (P>0.05) in values of temperature (38.721.23) °C, pulse rate (89.252.52), beats/minute (bpm), and respiratory rate (39.041.88) breaths/minute (bpm), when compared to values of negative calves; (38.481.56) ºC, (85.612.34) bpm, and (37.931.79) bpm, respectively (Figure 2). Regarding demographic risk factors, the findings showed a significant variation (P<0.05) between the values of the study groups (Table 1). For region factor, the prevalence of C. felis and the risk of infection increased insignificantly (P≤0.078) in rural (18.18% and 1.09, respectively) areas when compared to sub-urban (16.67% and 0.92, respectively) areas. Significantly, a higher rate of infection and risk was shown in calves aged <6 months (24.56% and 2, respectively) than in calves aged ≥6-12 months (12.33% and 0.5, respectively) (P≤0.03). Among groups of sex factors, females revealed a significant elevation (P≤0.015) in prevalence rate (20.79%) and exposure risk (3.02) to C. felis infection more than males (6.9% and 0.33, respectively).

Figure 1. Agarose gel electrophoresis of PCR products targeting the HSP70 gene Lane M: Ladder marker at 1500-100 bp; Lanes 1-23: Positive samples for C. felis; Lane P: Positive control; Lane N: Negative control

Figure 2. Results of clinical examination for positive and negative calves to C. felis

In this study, 5 local C. felis isolates were documented and named in the NCBI as Cryptosporidium felis Cattle No. 1, 2, 3, 4 and 5 isolates with the respective following accession numbers; MZ964600.1, MZ964601.1, MZ964602.1, MZ964603.1 and MZ964604.1. Multiple alignment analyses constructed using the NCBI-BLAST alignment tool showed a significant similarity (*) in HSP70 (Figure 3). Phylogenetic tree analysis based on HSP70 revealed that the genetic variant between the local C. felis isolates and NCBI-BLAST isolate was 0.0035-0.0005. An analysis of homology sequence identity between the local C. felis isolates and the NCBI-BLAST C. felis isolate detected a significant high identity between the local C. felis isolates and the Sweden C. felis (KP642069.1) isolate ranging 99.45% to 99.72% (Table 2, Figure 4).

Figure 3. Partial sequence of local C. felis isolates based on HSP70 gene

Figure 4. Phylogenetic tree analysis of the local and NCBI-BLAST C. felis isolates based on the HSP70 gene

4. Discussion

In the field, cryptosporidiosis can result in variable degrees of illness and poor health for both human and domestic animals due to direct and indirect contact with infected animals ( 7 ). Salyer, Silver ( 18 ) mentioned that infected companion animals, especially dogs and cats, risk transferring 60% of most infectious diseases. In this study, 17.69% of calves were positive for C. felis targeting the HSP70. However, several reports demonstrated infections of Cryptosporidium spp. ( 15 ), C. hominis ( 19 ), C. parvum ( 20 ), C. andersoni ( 21 ), C. bovis ( 22 ) and C. ryanae ( 15 ), little known are available about C. felis infection in cattle ( 23 ). Additionally, many studies have indicated significant variation in the prevalence of Cryptosporidium spp. between countries as C. bovis and C. andersoni are the almost detected species in African countries ( 24 ), while C. parvum is more prevalent in Australia ( 25 ) as well as the countries of Asia ( 26 ), North America ( 27 ) and Europe ( 28 ). However, the worldwide occurrence of cryptosporidiosis in cattle ranged 1.59-44.93% in African ( 29 ), 2.84-91.14% in American ( 30 ), 0.02-72.52% in Asian ( 31 ), and 2.56-55.6% in European ( 32 ) countries in addition to 0-71.85% in Australia ( 33 ) and 15.42-25.92% in New Zealand ( 34 ).

Based on our results, the HSP70 gene appeared as a highly sensitive tool for prevalence detection and genotyping of C. felis, which might be attributed to great levels of heterogeneity distribution over an entire sequence. Other studies have suggested other reasons for choosing of Cryptosporidium HSP70 gene, such as mRNA production that induces through simple heating to provide the desired inherent biological amplification; secondly, the HSP70 gene can be used as an indicator for oocyst viability, the half-life of HSP70 increases at least 10-fold upon heat shock, and easy to be identified from other related parasites which permitting for multiplexed detection ( 35 ).

We showed that values of the vital clinical parameters (temperature, pulse and respiratory rates) differed insignificantly, although all study animals were diarrheic. These findings indicate that C. felis infection in cattle does not result in a systemic reaction. Different studies have demonstrated that infections of Cryptosporidium spp. in cattle are commonly asymptomatic, particularly in adult animals ( 32 , 36 ). Rieux, Paraud ( 37 ) summarized that the prevalence rate of infection and the level of oocysts excretion vary greatly with distinct patterns according to the year. Other previously ( 38 ) and recently ( 39 ) reports found that C. parvum is calves' most commonly detected enteropathogenic cryptosporidial agent. de Graaf, Vanopdenbosch ( 40 ) mentioned that diarrhea in calves is a multifactorial disease governed by a wide range of factors related to the animal, condition of the environment and husbandry, and a variety of viruses, bacteria and protozoan parasites.

Statistical analysis for demographic risk factors in this study revealed a significant variation in values of age and sex groups but not in region groups. Calves of <6 months appeared at a higher rate and risk of C. felis infection than calves ages ≥ 6 months. These results were consistent with those observed by Bawm, Kyi ( 41 ) and Hussin, Khalaf ( 42 ). However, the high infection rate of cryptosporidiosis in calves aged <6 months might be attributed to poor immunity and high susceptibility to infection. Rieux, Paraud ( 37 ) showed that the age-related prevalence of cryptosporidiosis in calves is variable with systemically almost observation at the 2nd and 3rd weeks of life. In the USA, Lemeteil, Roussel ( 43 ) indicated that Cryptosporidium spp. is responsible for about 85-97% of Cryptosporidium infections in pre-weaned calves; but 1-4% of Cryptosporidium infections in post-weaned calves. Other studies demonstrated that the prevalence of specific Cryptosporidium species/subtypes changes among the different age groups of cattle ( 44 ). Some factors related to age or previous Cryptosporidium exposure might have delayed the onset of shedding in that study ( 45 ). Mamedova and Karanis ( 19 ) recorded that in the farm communities from the vertical belts, lowlands, foothills, and mountains, the younger cattle suffered higher consequences of Cryptosporidium infection than adult cattle. For sex factors, female calves showed a higher rate and risk of cryptosporidiosis than males. This result was similar to that reported by Mathew, David ( 46 ) and in contrast to those detected by Changizi, Salimi-Bejestani ( 47 ), who found no relationship between positive cases and sex of cattle, and to Ibrahim, Abdel-Ghany ( 48 ), who showed that Cryptosporidium prevalence was higher in males than females. Although the exact reason for the current observation is not apparent, we attributed this disparity between females and males to the usual practice of having a higher female-to-male ratio in a herd and the retention of female animals for breeding and milk production. The absence of significant differences in the prevalence of C. felis between calves of rural and suburban regions might presumably be due to direct contact between livestock, cattle, and cats.

In this study, genetic characterization of C. felis DNA revealed a significant association between the local C. felis isolates and NCBI-BLAST Sweden C. felis isolates (KP642069.1) at a 99.45 to 99.72% range of identity. In this Sweden study, the authors confirmed a case of human cryptosporidiosis is caused by the transmission of C. felis from a cat to a human ( 49 ). To a lesser extent, phylogenetic tree analysis also demonstrated a significant association between the local C. felis isolates and Australian C. felis isolates (AF221538.1) obtained from cats ( 50 ). The plausibility for passing C. felis from cats to calves was suggested in this study.

The nucleotide sequence of the HSP70 gene was helpful as a diagnostic tool for genotyping C. felis. However, additional studies are required to estimate the prevalence of infection and to detect the role of cats in the transmission of C. felis to animals and humans. The efficacy of the HSP70 gene and/or other genes in detecting Cryptosporidium spp. in fecal and environmental samples must be evaluated.

Authors' Contribution

G. J. K. A. and E. M. M. A. were responsible for collecting the samples of feces and for molecular testing, while H. A. J. G. was responsible for clinical evaluation of study animals, statistical analysis of results, and genetic sequence analysis. All authors participated equally in writing the final manuscript. No funds were received to complete this study.

Ethics

This study was approved by and performed under the license of the Scientific Committee of the College of Veterinary Medicine, Wasit University, Wasit province, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

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