Introduction

The bacterium A. phagocytophilum affects humans, horses, domestic ruminants, dogs, cats, and ticks and causes granulocytic anaplasmosis. Following a rearrangement of the families Rickettsiaceae and Anaplasmataceae under the order Ricketsiales, it replaced three species of granulocytic bacteria, Ehrlichia phagocytophila (ruminants), Ehrlichia equi (horses), and the agent of human granulocytic ehrlichiosis (1). Equine granulocytic anaplasmosis (EGA) was initially identified as a horse illness in California (2) and has subsequently spread to other regions of the United States and Europe South America (3,4). The disease's occurrence is influenced by the genetic variety of A. phagocytophilum, the host involved, and the vectors prevalent in a given location (5,6). Common symptoms include high fever, lethargy, partial anorexia, staggering or ataxia, distal limb edema, and hematological abnormalities such as thrombocytopenia, neutropenia, lymphopenia, and mild anemia (7). Clinical and laboratory symptoms (leukopenia, anemia, thrombocytopenia, and identification of intracytoplasmic inclusion bodies in leukocytes) are used to diagnose A. phagocytophilum in animals in the acute phase (3). Early-stage acute infection can be identified by the distinctive cytoplasmic inclusion bodies in neutrophils (8). A sensitive and specific PCR for A. phagocytophilum DNA detection in host blood has been created. PCR can detect A. phagocytophilum DNA in blood smears (9).

The current study aimed to determine the percentage of chronically infected carrier’s mules (Equus asinus × Equus caballus) by A. phagocytophilum. The molecular detection of A. phagocytophilum in mules using conventional PCR. DNA sequencing of A. phagocytophilum and Phylogenetic tree analysis.

Materials and methods

Ethical approve

Approval for this study was obtained from the committee of College of Veterinary Medicine/ University of Baghdad, Iraq. Number 39/PG on 7/1/2021.

Sampling

50 blood samples from 50 mules were taken aseptically. Blood was drawn from the animals through jugular vein punctures in tubes containing the anticoagulant (EDTA). These samples were used for detecting the presence of A. phagocytophilum by making thin film slides of blood smears. Positive and negative samples were submitted to DNA extraction molecular characterization by using conventional Nested PCR for detection of A. phagocytophilum. The amplified DNA was sequenced to confirm the detection of local A. phagocytophilum.

DNA extraction

DNA was isolated from blood samples according to the manufacturer's instructions using the gSYAN DNA kit extraction kit (Frozen Blood) Geneaid (Taiwan) using a Nanodrop spectrophotometer (THERMO. USA) for checking DNA quantity by measuring absorbance at (260/280 nm).

PCR Reaction

The Green PCR Master Kit was used to create the PCR master mix prepared according to the manufacturer's recommendations. PCR thermocycler settings were accomplished using a typical PCR thermocycler system: 5 minutes of denaturation at 95°C, denaturation at 95C° for 30 sec, annealing at 55 C°30sec, extension at 72C°1min-5 minutes at 72 C°, final hold is at 4 C°. Nested PCR findings were examined using agarose gel electrophoresis. Agarose gel electrophoresis was used with red safe dye with a specific ladder marker (100-2000bp) to specify the molecular weight of amplified genes sequences which appear as bands. Multiple sequence alignment analysis of the missing Msp4 genes based on ClustalW alignment analysis was used in conjunction with DNA sequencing using Molecular Evolutionary Genetics Analysis version 6.0 (Mega 6.0). The Maximum Composite Likelihood approach and the UPGMA algorithm on phylogenetic trees were used to calculate the evolutionary distances (Table 1).

Table 1: Primers and PCR scripts for identifying A. phagocytophilum

PCR program: I: 35 time (95ºC30sec, 55ºC30sec, 72ºC1min). II: 35 time (95ºC30sec, 55ºC30sec, 72ºC1min).1: 35 time (95ºC30sec, 54ºC30sec, 72ºC1min). 2: 35 time (95ºC30sec, 55ºC30sec, 72ºC1min).

Statistical analysis

The data from the current study were statistically analyzed using the Statistical Package for Social Sciences version 28. The chi-square test was done to determine the relationship between the variable percentages. Descriptive statistics and an independent t-test were utilized to examine clinical and hematological variables to compare the mains of two groups at a 95% confidence interval (p0.05). Test results were considered statistically significant if their P value was less than 0.05 at the significance level (13).

Results

The infection rate in blood smear

All of the mules in the study were clinically assessed. The infection was persistent in those animals, and varied clinical symptoms were detected in those infected with A. phagocytophilum, including emaciation and a pale mucosal membrane in the infected mules. Temperature, respiration, and heart rate were average. varied clinical signs in infected animals, and some infected animals had no clinical indications. This study, A. phagocytophilum was identified by looking for the organisms (morula) in buffy coat smears and blood samples. Blood smear testing for morula in granulocyte cytoplasm significantly predicts a diagnosis. For the first time, A. phagocytophilum was found in the blood of anemic, underperforming, and losing weight mules. For the first time in Iraq, blood samples from mules were examined for A. phagocytophilum (Figure 1 and Table 2)

Figure 1: Shows the morula in the blood of mules.

Table 2: Distribution of positive blood smear in mules

The percentage of positive cases of blood smear in microscopic examination was 4(50%) out of 8 (100%) positive samples by PCR test out of 50 mules. The blood smear result in infected mules was significant at p≤ 0.001.

Molecular detection

Nested PCR was used as a detection technique for A. phagocytophilum studied gens (16 SrRNA and mgsp4) to visualize the positive amplified gene sequences (Figures 2 and 3 also Tables 3 and 4).

Figure 2: The results of the 16Sr RNA gene in A. phagocytophilum blood samples are shown. Lanes (1-8) showed only Positive samples at (546bp) Nested PCR product where M: marker.

Table 3: Distribution of positive 16S RNA gene in mules

This table shows the positive samples in the nested PCR test of the 16S RNA gene in A. phagocytophilum from mule^'s blood samples. The percentage of infected animals was 8 (16%) and 42 were non-infected out of 50 mules. The result of the nested PCR test of the 16SrRNA gene in A. phagocytophilum in infected mules was significant at p≤ 0.001.

Figure 3: The msp4 specific gene in A. phagocytophilum from mule by Nested PCR. Lanes 1-8 and M represent positive samples and DNA markers (100 - 2000bp) respectively. The product size was 381bp.

Table 4: Distribution of msp4 gene positive cases in mules

This table shows the positive samples in nested PCR test of the msp4 gene in A. phagocytophilum from mule^'s blood samples and the percentage of infected animals was 7 (14%) and 43(86%) were non-infected out of 50 mules. The result of nested PCR test of the msp4gene in A. phagocytophilum in infected mules was significant at p≤ 0.001.

DNA Sequence results

The genetic relationship study of the Msp4 gene in A. phagocytophilum isolates and NCBI-Genbank relatedness A. phagocytophilum country isolates was performed using DNA sequencing. The examination of the phylogenetic tree (genetic connection) revealed that A. phagocytophilum mule isolates showed a close relation to NCBI-BLAST A. phagocytophilum Hungary isolates at total genetic changes 0.1%. The homology sequence identity between A. phagocytophilum mule isolates and NCBI-Genbank related A. phagocytophilum Hungary isolate showed a genetic homology sequence identity range 99.21%. furthermore, the phylogenetic tree similarity was 96%. Finally, A. phagocytophilum mule isolates were submitted to NCBI Genbank and identified by accession numbers OP244689.1-OP244690.1 (Figure 4 and Table 5).

Figure 4: Msp4 gene partial sequence analysis in local A. phagocytophilum mule isolates used for genetic connection analyses. At total genetic alterations (0.1%), the A. phagocytophilum mule isolates were found to be closely related to the NCBI-BLAST A. phagocytophilum isolate Hungary isolate.

Table 5: The proportion of NCBI-BLAST Homology Sequence identity between local A. phagocytophilum mule isolates and NCBI-BLAST closed genetic related A. phagocytophilum country isolates

Phylogenetic tree study based on the partial sequence of the (Msp4) gene in a local A. phagocytophilum mule isolate used for genetic connection analysis. The phylogenetic tree was built in MEGA 6.0 using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA tree). At total genomic alterations 0.1%, the A. phagocytophilum mule isolate was closely related to the NCBI-BLAST A. phagocytophilum isolate Hungary isolate.

Discussion

1. phagocytophilum causes granulocytic ehrlichiosis in various domesticated mammals, including equids. A. phagocytophilum is endemic or potentially endemic in 42 countries of the world. This has been detected throughout Europe, America (North and South), Asia (Pakistan, India, Korea, and Japan) and Africa (14-21). Human seroprevalence in disease endemic area of Wisconsin and New York (USA) is 15-36%, whereas seroprevalence in Europe range from 1 and 20% depending upon immunity, tick exposure, and age of the patients (22). Majority of the human cases of infection in USA occur in June-July.

 This study showed that A. phagocytophilum infects mules in the north of Iraq. The mules in this study did not show clinical symptoms, and This outcome was consistent with what others had reported (23,24). This could be due to several factors, such as infection phase (acute or carrier), immunological status, age, infective dose, environment, and management. This makes them chronically infected carriers and keeps their clinical symptoms under control (25).

In this investigation, A. phagocytophilum organisms were discovered microscopically in Iraqi mules, the microscopic examination of blood smears is an easy, rapid, and cheap field test that used in the diagnosis of A. phagocytophilum (26,27). The anaplasma's intracellular replication results in forming these morulae. The morula observed in the cytoplasmic vacuoles of white blood cells specially neutrophils in blood smears, is considered diagnostic for ehrlichiosis (11), and the results agreed with another study Saleem et al. (28) on mules in Pakistan, Torina et al. (29) on donkeys in Sicily- Italy, Naranjo et al. (30) in Spain, Yousefi et al. (31) on dogs in Iran, Atif (32) and Taylor et al. (33). This method has low sensitivity, in cases of chronic, subclinical, or persistent infection. Therefore, the results of the microscopic examination method should be confirmed using more sensitive and accurate techniques such molecular techniques (34).

The molecular characterization of A. phagocytophilum and the use of 16SrRNA gen and the results of this study agree with previous studies conducted by Saleem et al. (28), Torina et al. (29), Naranjo et al. (30), Yousefi et al. (31) for the detection of A. phagocytophilum in donkeys and other animals. The diagnosis of A. phagocytophilum using the msp4 gene and results agreed with the study Saleem et al. (28), Torina et al. (29), Naranjo et al (30). MSP4 is expressed on the outer membrane of A. phagocytophilum. MSP4 is thought to be involved in host-pathogen interaction and may evolve more rapidly than other nuclear gene proteins, resulting in host-specific characteristics due to selective pressures exerted by host immune systems, resulting in high sequence heterogeneity among A. phagocytophilum strains in this particular gene (10,11). Separate clustering in ruminants is another example of evolution connected to host sensitivity and geographical distribution of this creature (11).

The ability to distinguish A. phagocytophilum samples based on their mammalian host of origin suggested that msp4 sequences might be employed for coevolutionary research (11). The msp4 sequences of the MRK isolate, which was initially isolated from a horse in California, and the Italian strains from donkeys were identical (11). phylogenetic analysis of the msp4 sequences differentiated between strains of A. phagocytophilum from humans, dogs, and horses from those obtained from ruminants (35). This study and other studies have shown that A. phagocytophilum and many blood parasites infects mules and many animals like small and large ruminants and the infection spreads in the north and middle of Iraq (35).

Conclusion

It can be concluded from the result that the chronic form of the disease was prevalent, but the acute form was not recorded. A. phagocytophilum was recorded for the first time in the mules in Iraq. The use of specific primers for nested PCR tests was particular and sensitive to microscopic examination.

Acknowledgment               

The College of Veterinary Medicine at the University of Baghdad is commended for providing the author with all the resources needed to complete this work.

Conflict of interest

There is no conflict of interest.

Editorial board note

Saleem A. Hasso, the editor of the Iraqi Journal of Veterinary Sciences, did not participate in any stage of the decision-making process for this article.