The Affects of Genital Myiasis on the Diversity of Vaginal Flora in Female Bactrian Camels

Background: One of the most important diseases that affect the reproductive organs of Bactrian camels is called Genital Myiasis. It can cause serious mechanical damage to the vaginal tissue of female Bactrian camels. The accumulation of bacteria in the vagina of female camels can affect their health and reproductive ability. The effect of this damage is commonly found in the vaginal ora and vaginal mucosal immune system. Therefore, this research is a study of the diversity of the vaginal ora and the differences between healthy Bactrian camels and those suffering from Genital Myiasis. Results: Vaginal microbiota samples were collected from two groups of female Bactrian camels of the same age. Illumina Miseq was used to sequence V3-V4 hypervariable genes of 16S rRNA in the samples, and the results showed that the vaginal microora of the infected camel had a signicantly greater OTU value. According to the Alpha diversity index and the level of vaginal pH, the diversity index of the infected camel ora were higher than that of the normal camel ora, and the pH were lower than that of the normal camel ora (P=0.006). There was no signicant difference between the two groups in the abundance of dominant genera of Bactrian camel vaginal (P (cid:0) 0.05), indicating that the structure of dominant ora of Bactrian camel vagina had a certain stability. Conclusions: Overall this comparison revealed the differences and similarities between vaginal ora Bactrian camels in various health states. In addition, this data provides a reference point for understanding the types of bacteria that cause genital myiasis that damages healthy development of Bactrian camels.


Introduction
Bactrian camel is one of the unique domestic animals to China. It mainly lives in the hot and arid regions of the Gobi and Desert area in northwestern China. It is known as the "boat of the desert" (Mengli et al., 2006;Ji et al., 2010;Zhichao et al., 2016). For a long time,the development of the bactrian camels breeding industry has been hampered by Genital Myiasis which ha s brought serious economic losses to local herders.
The Genital Myiasis of Bactrian camels is a serious parasitic disease. Larvae of Wohlfahrtia Magni ca (Schiner, 1862) parasitize around the perineal and vaginal region of Bactrian camels and are responsible for a severe obligatory traumatic myiasis (Robbins et al., 2010). Genital Myiasis has a distinct seasonality which occurs in May-September of each summer and autumn (Kunichkin et al., 1981;Lungu et al., 1985;Hadani et al., 1989;Valentin et al., 1997). Clinical symptoms manifest as severe mechanical damage in a variety of ways to the affected tissue and mucosal sites harmful affects, such as, local in ammation, anxiety and anorexia are some of the symptoms in diseased camels (Valentin et al., 1997;Giangaspero et al., 2011;Sazmand et al., 2017). Through long-term experimental observation we found that the diseased camel's vaginal wound was exposed to the external environment but was rarely infected and purulent.
When the larvae of Wohlfahrtia Magni ca was detached from the host, 94.5% of the diseased camel wounds spontaneously recovered (Schumann et al., 1976). In addition, other important elements comprise the vaginal microenvironment.
The vaginal mucosa in healthy animal is colonized by an equilibrated and dynamic composition of aerobic, facultative anaerobic and obligate anaerobic microbes (Srinivasan M et al., 2021 antibiotics or antifungal drugs were used systematically within one month. Furthermore, sterile procedures were applied to the sampling area. Routine sterile operations were used before each sampling and strictly followed. In addition, the procedural steps to strictly ensure an aseptic open the female camel's vaginal and rolled 5 times, along the vaginal wall to wipe the vaginal secretions. Then they were quickly placed in a sterile 5ml cryotube. Lastly, the sample was labeled and quickly stored in a liquid nitrogen or in a -80℃ refrigerator and used to extract the 16S rRNA gene. Shortly afterwards, the pH of each sample was measured using an UltraBasic pH meter (Denver Instruments Arvada CO United States).

Bacterial DNA Isolation
The thawed sample was centrifuged at 10000r for 10 minutes to collect bacterial cells and the supernatant was discarded. The total DNA of the sample was extracted using the vaginal swab genomic DNA kit (Qiagen QIAamp DNA Mini Kit) and the speci c steps were referred to the instructions. The DNA was extracted and stored in a refrigerator at -20 °C. The DNA extraction quality was measured by 0.8% agarose gel electrophoresis and the DNA was quanti ed by an ultraviolet spectrophotometer.

Sequencing of 16S rRNA
In combination with the uorescence quanti cation results, each sample was mixed in a corresponding ratio according to the sequencing amount requirement of each sample. The processed samples were sent to Beijing WEISHENGTAI Co. Ltd. for double-ended 2×300 bp sequencing (Paired-end) through the Illumina HiSeq 2000 platform.

Sequence Read Processing and Statistical Analysis
Basic statistical analysis was performed using SPSS Statistics 20.0 statistical analysis software. Two pairs of comparisons of the measured data were taken, in accordance with the normal distribution. They were performed using two independent samples test P < 0.05. Therefore, they were was considered statistically signi cant.

Vaginal pH
The vaginal pH of all 23 female Bactrian camels was measured. The results showed that the vaginal PH range of the healthy group of bactrian camels ranged from 7.47 to 8.23 with an average of 7.85 ± 0.13. The vaginal PH of the diseased group was in the range of 7.18-7.61. Also, the average was between 7.41 ± 0.11. Such that the vaginal PH of bactrian camels was signi cantly different between the normal group and the group that was ill (p = 0.006). Also, the vaginal PH of the group that was ill was lower than that of the normal group.

Sequencing Information
After optimization of quality control and chimera removal, a total of 1644139 reads were obtained for all 23 samples. That had with an average of 71484 reads per sample (Table 1). Samples from the group that was ill were taken and received a total of 744455 reads, with an average of 77446±11214 reads per sample. The normal group samples received a total of 899684 reads with an average of 69206 ± 11047 reads per sample. The results showed that the statistically signi cant differences in the number of optimized sequences, obtained between the two groups were not signi cant (P>0.05).

Alpha-and Beta-Diversity
The sequence obtained above was subjected to merging, and revealed the OTU division by 97% sequence similarity. Also, the OTU having abundance value lower than 0.001% of the total. Also, the sample sequencing amount was removed (Bokulich et al., 2013). A total of 1845 OUTs were detected with an average of 1689. Also, 1267 OUTs were detected in the group that was ill. In addition, the normal environment vagina for each was maintained with 1111 OTUs shared between various vagina environments ( Fig. 1).
Alpha-diversity was measured and observed using OTU Chao1, ACE, simpson and Shannon Diversity Index. The conclusive analysis presented in Table1. No signi cant differences existed in alpha-diversity between the normal samples of female Bactrian camels those that were ill and vaginal bacterial observed OTU Chao1 ACE simpson index and Shannon's Diversity Index (p>0.05). But the illness bactrian camels vagina had a signi cantly greater number of OTU than did the normal bactrian camels vagina increased richness as measured by Chao1 and ACE and greater diversity as measured by Shannon's Diversity Index and the Simpson Index all of which are presented in Table 1. Beta-diversity was also analyzed to examine differences in microbial communities between samples.
Using an OTU-centric approach PCoA matrices were employed using weighted and unweighted UniFrac distance matrices to compare the phylogenetic divergence among the OTU between samples from ill camels and healthy camel vaginal samples (Fig. 2). The results showed that the clustering of subsets of healthy camel vaginal samples was more closely clustered in the weighted and unweighted UniFrac distance matrix. In addition, ANOSIM analysis showed that there was a signi cant difference between the vaginal samples of ill female camels and normal camels (P=0.033). R statistics showed that the difference between the groups was signi cantly greater than within groups (R=0.1483) and the grouping effect was evidently well done.

Taxonomic composition analysis
According to the results of OTU classi cation and classi cation status identi cation, the dominant vaginal ora and average relative abundance of Bactrian camels in the normal group and the illness group were respectively identi ed at the phylum level: Firmicutes ( Using the visualization tool GraPhlAn (Asnicar et al., 2015) to build a hierarchical tree of the composition of the sample population at each classi cation level (Fig. 3). More information is evident. While each classi cation unit was distinguished by different colors and their distribution in abundance was also re ected by the node size.
Using the Mothur software, called the statistical algorithm of Metastats (http://metastats.cbcb.umd.edu/) (White et al., 2009). We were able to determine the overall classi cation level of all classi cation units in the sample population. The difference of sequence quantity (i.e. absolute abundance) between each taxon at phylum and genus level was analyzed and compared (pin-wise). We found that there were 4 classi cation units with signi cant differences in gate levels (Fig. 4) namely: SR1 (p=0.030 q=0.120); Planctomycetes (p=0.030 q=0.120); Gemmatimonadetes (p= 0.041 q = 0.120); Elusimicrobia (p = 0.048 q = 0.120). There are 51 taxonomies with signi cant differences in levels (  (Rooks, 2016). This study is an analysis of basic research that was conducted. By comparing the differences in the structure and diversity of normal Bactrian camels and with camels that were ill, we were able to analyze the role of the vaginal microecosystem of Bactrian camels, in their immunity and recovery stages, after their infections of vaginal myiasis. More understanding of these stages may provide a new approach for the prevention and treatment of genital myiasis of Bactrian camels, that result in positive results for clinical treatment of genital myiasis.
In this study the bacterial phyla with the highest abundance identi ed in the two groups of Bactrian camels' vaginal samples were Firmicutes Proteobacteria Fuso bacterium and Bacteroides. These phyla are representative of the most common phyla found in many environments especially in host-microbiome relationships. Studies have shown that the proportions and relative abundance of these gates are related to changes in host physiology. Therefore when we performed ANOSIM analysis on the samples we found that even if there were differences among different individuals in the same group the difference was obviously smaller than the difference between the groups. We think this difference is reasonable.
As a natural channel, the vaginal ora is susceptible to environmental microbes. The increase of the diversity and richness of the bacterial community, in the vagina of the diseased camel can be explained by the fact that its vulva is affected by y maggots, which causes swelling and deformation and cannot be completely closed, while a large number of external bacteria enter the vagina. However, the taxonomic composition analysis of bactrian camels showed that there was no signi cant difference in the overall structure of its vaginal ora, indicating that the vaginal microecology of Bactrian camels had certain stability. In addition immunomodulatory symbionts induce speci c self-targeted responses that indirectly regulate immune responses to surrounding microorganisms (Ost and Round, 2018  In addition, this study also analyzed the microbiome of the Bactrian camels vagina, to determine the relationship between the presence (or absence) of certain microbiota and the vaginal mucosal immune system. Overall this study will be used to document changes in the diversity of vaginal microbiota in healthy camels and also for that that are suffering from vaginal myiasis to identify unique microbes that may be involved in the vaginal mucosal the immunity. And it may help determine changes in the microbiome associated with the immune regulation, that may be bene cial and positive, throughout the pathological cycle.

Declarations
Ethics approval and consent to participate The sampling process did not cause any damage to the vaginal mucosa of Bactrian camel.In this experiment, the breeding environment was in compliance with the standards relevant to an ordinary animal laboratory facility in China National Standard "Laboratory animal environment and facilities" (GB14925-2010). The feeding of and the experimental operations on animals were in accordance with the animal welfare requirements.All experimental procedures were approved by the Animal Protection and Use Committee of Inner Mongolia Agricultural University and strictly followed animal welfare and ethical guidelines.

Consent for publication
Not applicable Availability of data and materials We have submitted raw data through supplementary materials.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by The National Natural Science Foundation of China (Grant No. NSFC 31360591). Authors' contributions EEDMT developed a research program and funded it; ZLK carried out the experiment, analysis and article writing; BH and HBX participated in the data analysis; ADD helped write the article, and the other authors participated in the sample collection.All authors read and approved the nal manuscript. Principal coordinate analysis of vaginal samples from ill female camels and normal female camels, using UniFrac unweighted (A) and weighted (B) metrics. Vaginas sample from ill female camels (n=10) are represented by red squares and Vagina samples from normal camels(n=13) are represented by blue circles.

Figure 3
Sample overall classi cation level tree diagram based on GraPhlAn Note: The classi cation level tree shows the hierarchical relationship of all classi cation units (represented by nodes) from the gate to the genus (from the inner circle to the outer circle) in the sample population. The node size corresponds to the average relative abundance of the classi cation unit. The top 20 units of relative abundance will also be identi ed by letters in the gure (from door to genus in order from outer layer to inner layer) and the shadow color on the letter is the same as the corresponding node color.

Figure 4
Abundance distribution of phylum-level taxa, with signi cant differences between sample groups Figure 5 Abundance distribution of the top 20 taxa with signi cant differences in genus levels Note: The abscissa in the gure is the taxonomic unit, that shows a signi cant difference and the ordinate is the sequence quantity of each taxon in each sample group. The border of the gure represents the Interquartile range (IQR), the horizontal line represents the median value, and the upper and lower tentacles represent the 1.5 times IQR range, except the upper and lower quartiles. Also the symbol "•" indicates the extreme value exceeding the range.