Genital Infection and Associated Pathology in Red Sokoto and West African Dwarf Does in Makurdi ()
1. Introduction
Goat plays an important role in developing countries because of its meat and milk production [1] and contributes to health and nutrition. Their potentially high reproductive rates and production efficiency has lead to an increased proliferation of small and medium scale goat farmers in Nigeria [2]. However, regular and successful reproduction is the key to profitable goat production. Poor fertility is a common problem in ruminants and it has been related to diseases involving different parts of the female genital tract [3]. This can be a complication of various infectious diseases that localize in the reproductive organ of goats with or without specific morphological defects [4] [5]. Such lesions may be part of a systemic disease or initiated as reproductive organ disturbances following breeding or obstetric interventions [6] [7] [8] [9]. Many of these reproductive pathologies are associated with diseases that lower the overall reproductive performance of the animals or cause fetal mortality and abortions [10].
The number of bacteria colonizing the reproductive tract is important determinants of uterine infections [9] [11]. Genital microflora are usually harmless until presence of predisposing factors such as trauma, systemic infection [12], lowered immune status during stress and cystic ovarian disease [13]. Genital microflora may act as opportunistic bacteria to cause genital infection that usually leads to reproductive failure in ruminants [4] [5] [14] [15] [16]. Varieties of bacteria have been isolated from the genitalia of the does and were shown to be associated with disease conditions of the genitalia [17] [18].
Various investigations have been carried-out and valuable suggestions made on sensitivity of bacterial species to different antimicrobial agents [19] [20] [21]. Antimicrobial agents are commonly used in the management of reproductive failures in livestock [22]. An insight on the microbes colonizing genital tract and their antimicrobial susceptibility will therefore demystify management of genital tract infections and abnormalities in does. The objective of this study therefore was to investigate and compare genital tract microbes and associated genital disorders in Red Sokoto and West African Dwarf does and performs antimicrobial sensitivity for an effective management of reproductive abnormalities in small ruminants.
2. Materials and Methods
2.1. Study Area, Animal and Sample Collection
The study was carried out in Makurdi, Benue state, north-central part of Nigeria on longitude 8.35˚E and latitude 7.44˚N with a radius of 16 km. The climate is tropical and the vegetation is predominantly guinea savannah with an annual rainfall of 1090 mm. This area is defined by two seasons, the rainy season (April to October) and dry season (November to March) with an average relative humidity of 26.3% to 71.3% throughout the year. The atmospheric temperature ranges from 27.38˚C to 34.09˚C [23].
Sample size was determined at 95% confidence level by using the formula of Thrusfield (1997):
where N = sample size, Z = appropriate value for the standard normal deviation for the desired confidence = 1.96, p = prevalence, q = 1 – p, d = level of significance (0.05) with assumption that 70% of the genitalia (non pregnant) of RS and WAD does will be infected with bacteria. A total of 100 (50 each from RS and WAD does) were from Wurukum and International cattle market abattoirs in Makurdi, respectively, from April to June, 2016. The genital tracts were collected twice weekly after evisceration and transferred on ice in clean labeled polyethelyne bags to the pathology laboratory. Ethical approval for this study was obtained from the College of Veterinary Medicine, Federal University of Agriculture Makurdi (FUAM).
2.2. Collection of Samples for Bacteriological Studies
Genital organ swabs from RS (44) and WAD (38) does were collected as per standard protocols described by [24] and [25]. Lesions were examined by their size, consistency, colour, shape, smell or location as described by [26]. Each reproductive tract was opened by cutting with a pair of sterilized scissors, starting from the vulva, into the vagina through the cervix and uterine body, into each horn, saphinx and finally the ovaries. Sterile cotton swab sticks (Nicecare®, Nigeria) was rolled over lesion-bearing and normal portions of the genital tracts accordingly. The genital swap samples were transported on ice in cold boxes to Central Diagnostic Laboratory, National Veterinary Research Institute (NVRI), Vom, in labeled Bijou bottles containing 2 ml nutrient broth.
2.3. Bacterial Culture, Isolation and Identification
Each swab sample was inoculated on to conventional culture media Blood agar (BA), MacConkey agar (MCA) and Nutrient agar (Oxoid, Basingstoke, UK), respectively, for the isolation and purification of bacteria. The media were prepared according to the manufacturer’s instruction. Sterile wire loop was used to streak out the inoculums to allow spread of bacteria on the media to produce discrete colonies. Discrete colonies on the culture media were examined and identified based upon colony morphology and pigment characteristics; a colony of each morphologic type was purified by a repeated subculture at 37˚C for 24 hours on to MCA, BA and Eosin methylene blue (EMB) [24]. The purified bacteria cultures were identified based upon reaction to Gram stain and microscopic characteristics (×100 lenses) as described by [27] and bio-chemical tests as described by [28].
2.4. Antibiotic Sensitivity Test
The antibiotic sensitivity was determined according to Kirby-Bauer (disc diffusion) method described by [29]. The [30] criteria was set to record the level of sensitivity. The diameter of the zone of inhibition was measured (mm): <10 mm = Resistant, >10 mm = Moderately sensitive and ≥15 mm = Sensitive.
2.5. Data Analysis
Descriptive statistics was used to represent the data generated. The distribution of the bacterial isolates was estimated as simple percentages. Relative risks (RR) for an infection with bacteria were analyzed. The pattern of bacterial isolation and rate of infection of genital tracts with bacteria was determined and expressed in percentage. All data analysis was subjected to Graphpad Prism Statistical Software [31] GraphPad Software Inc. (2016). GraphPad Prism Version 7.03. P-value was considered significant at <0.05.
3. Results
3.1. Distribution of Bacterial Isolates from Genital Tracts of RS and WAD Does
Seven genera of bacteria were identified from RS which includes Escherichia coli, Staphylococcus spp, Pseudomonas spp, Klebsiella spp, Proteus spp, Enterobacter spp and Citrobacter spp while six genera of bacteria were identified from WAD genitalia which includes Escherichia coli, Staphylococcus spp, Pseudomonas spp, Klebsiella spp, Proteus spp and Citrobacter spp (Table 1).
A total of 113 bacterial isolates were recovered from genital tracts of RS and WAD does, respectively (Table 2). Of the 59 bacterial isolates identified from RS (n = 44), 42 were recovered from the uterus: Escherichia coli 20 (46.0%), Pseudomonas spp 15 (34.0%), Klebsiella spp 3 (7.0%), Staphylococcus spp 2 (5.0%), Proteus spp 1 (2.3%) and Citrobacter spp 1 (2.3%). Vaginal cultures yielded 16 isolates: Escherichia coli 7 (16.0%), Pseudomonas spp 4 (9.0%), Klebsiella spp 2 (5.0%), Enterobacter spp 2 (5.0%) and Proteus spp 1 (2.3%). 1 (2.3%) isolate was recovered from the cervix while no bacteria were isolated from the salphinx (Table 2).
In the WAD (n = 38), 54 bacterial isolates were identified, out of which 37 were recovered from the uterus (Table 2): Escherichia coli 16 (42.1%), Pseudomonas spp 11 (28.0%), Klebsiella spp 2 (5.3%), Proteus spp 3 (8.0%), Staphylococcus spp 3 (8.0%) and Citrobacter spp 2 (5.3%). The vaginal cultures yielded 14 isolates: Escherichia coli 7 (18.4%), Pseudomonas spp 4 (11.0%), Klebsiella spp 2 (5.3%) and Proteus spp 1 (3.0%). 3 isolates were recovered from the salphinx which includes Escherichia coli 1(3.0%), Pseudomonas spp1 (3.0%) and Klebsiella spp 1 (3.0%) while no bacteria were isolated from the cervix (Table 2).
3.2. Relative Risk (RR) Analysis
The relative risk for infection (RR) of the uterus with Klebsiella spp (1.30, CI: 0.2282 to 7.352, P = 0.7692) was higher in RS compared to WAD, while the RR for Escherichia coli (1.08, CI: 0.6588 to 1.769, P = 0.7606) and Pseudomonas spp (1.18, CI: 0.6174 to 2.237, P = 0.6177) were lower in RS compared to WAD. However, the RR for infection of the vagina with Escherichia coli (0.86, CI: 0.3328 to 2.241, P = 0.7631), Pseudomonas spp (0.86, CI: 0.2314 to 3.222, P = 0.8271) and Klebsiella spp (0.86, CI: 0.2282 to 7.692, P = 0.7692) were the same for RS and WAD. The RR for infection of the vagina and uterus between RS and WAD was not statistically significant (P > 0.05). The results are presented in Table 2.
3.3. Rate of Genital Infection with Bacterial Isolates in RS and WAD Does
The rate of genital infection with bacterial isolates in RS and WAD does is shown in Table 3. Both RS and WAD genital organs were associated with Escherichia coli (63.4%) followed by Pseudomonas spp (31%) and Klebsiella spp (12.2%).
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Table 1. Bacterial isolates from genital tracts of RS and WAD does.
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Table 2. Distribution of bacterial isolates from different parts of the genital tracts of RS and WAD does.
(n = 44, n = 38 is the total number of swabs collected from genital organs of RS and WAD respectively). RR is the relative risk for an infection with bacteria in RS compared to WAD does. P = Probability for infection (P > 0.05) is not significant. CI = Confidence interval. RS = Red Sokoto doe, WAD = West African Dwarf doe.
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Table 3. Infection rate of bacterial isolates from genital tracts of RS and WAD does.
P > 0.05. (n = 44, n = 38 is the number of swabs collected from genital tracts of RS and WAD respectively). RS = Red Sokoto doe. WAD = West African Dwarf doe.
3.4. Pattern of Bacteria Isolation from Genital Tracts of RS and WAD Does
A total of 30 isolation patterns in RS and 23 patterns in WAD does were recorded from apparently normal genital tracts with Escherichia coli being the predominant while 12 isolation patterns in RS and 14 patterns in WAD does, respectively, were recorded from genital tracts with abnormalities with Escherichia coli being the predominant (Table 4).
3.5. Bacterial Isolates from Genital Tract Lesions in RS and WAD Does
Bacterial isolates from the genital tract of RS and WAD does associated with lesions are presented in Table 5. Escherichia coli, Proteus spp and Staphylococcus spp were isolated from cases of endometritis and uterine congestions while Escherichia coli and Staphylcoccus spp were isolated from pyometra, Escherichia coli and Proteus spp were isolated from case of uterine melanosis. Escherichia coli, Staphylococcus spp and Pseudomonas spp were isolated from postparturient metritis, while Pseudomonas spp and Citrobacter spp isolated from postparturient emphysematous metritis. Escherichia coli, Proteus spp and Pseudomonas spp were isolated from mucometra. Klebsiella spp and Pseudomonas spp were isolated from case of salphingitis. While Escherichia coli was isolated from acute cervicitis, Proteus spp was isolated from hemorrhagic necrotizing cervicitis.
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Table 4. Pattern of bacterial isolation from genital tracts of RS and WAD does.
RS (n = 31), WAD (n = 25) is the number of apparently normal genital tracts of RS and WAD, respectively. RS (n = 13), WAD (n = 13) represents number of genital tracts of RS and WAD with pathology, respectively.
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Table 5. Bacteria isolated from genital tract lesions in RS and WAD does.
3.6. Antimicrobial Susceptibility Test
The bacterial isolates (Gram –ve) show high susceptibility (77% - 94%) to Levofloxacin, Pefloxacin, Ciprofloxacin, Ofloxacin, Amoxyl and Gentamycin. The susceptibility to Amoxycillin clavulanate, Streptomycin and Ceporex were moderate (50% - 57%) while low rate was recorded for Ampiclox (34%) (Table 6). Staphylococcus spp showed high susceptibility (80% - 100%) to Levofloxacin, Ciprofloxacin and Amoxyl. The susceptibility to Norfloxacin, streptomycin, Gentamycin, Rifampicinand ampiclox were moderate (40% - 60%) while Chloramphenicol and Erythromycin showed low susceptibility (20%) (Table 7).
4. Discussion
Bacteria colonizing the vagina and uterus are likely to cause reproductive failure in domestic ruminants and are important determinants of uterine infections [9] [11] [32] [33]. Vaginal bacteria get access into the uterus during the peripartum period leading to metritis and endometritis and subsequent reduction in the reproductive capacities of these animals [4].
Out of the vaginal bacterial isolates recovered from RS and WAD in this study E. coli and Pseudomonas spp were the most common. The isolation rate of E. coli in RS and WAD (16% and 18.4%), respectively, was higher compared to the rate of for Pseudomonas spp similar to previous report [34] [35] [36] [37]. Klebsiella spp, Enterobacter spp and Proteus spp were also isolated with the later having the lowest isolation rate of 2.3%.
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Table 6. Frequency of antibiotic susceptibility of bacterial isolates from genital tracts of RS and WAD does.
n = total number of Gram –ve bacteria isolated from genital tracts of RS and WAD does.
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Table 7. Frequency of antibiotic susceptibility of Staphylococcus spp from genital tracts of RS and WAD does.
n = total number of Staphylococcus spp isolated from genital tracts of RS and WAD.
The uterine bacterial isolates in the RS and WAD in this study are similar to those observed in the vagina. This was similarly reported previously [8] [9] [34] [36] [37]. E. coli was the common uterine bacterial isolate with an isolation rate of 46% and 42.1% in RS and WAD, respectively. Citrobacter spp and Staphylococcus spp were isolated while Enterobacter spp was not isolated. Bacteria isolated from the uterus were more than other portions of the genital tracts of both RS and WAD in this study similar to reports of [15] [38] [39]. Klebsiella spp and Pseudomonas spp were isolated from the salphinx in WAD while none was recovered in the RS as previously reported [34]. These bacteria may be present as opportunist from the uterus as a result of ascending infections during clearance of bacteria from the uterine lumen [8] [36]. E. coli was recovered in the cervix only in the RS in this study.
The overall infection rate (63.4%) of the genital tract with E. coli in RS and WAD was higher than other bacterial isolates in this study. This agrees with the report of [37] that the overall weight of infection with E. coli (79%) was more than other isolates put together in cows and camels. However, in this study, the rate of infection with E. coli was higher in the uterus compared to other parts of the genital tracts contrary to earlier findings of [36] [37] who reported high rate of E. coli in the vagina compared to the uterus. The importance of E. coli as cause of genital disorders in animals cannot be ignored because it has been frequently reported as the most common isolate of the genital tract [34] [35] [36] [37].
Bacterial isolates from normal and pathological cases in genital tracts of ewes have been reported [18] [40]. Evidence implicating bacterial infections as causes of endometritis has been reported, and a variety of these bacterial species have been recovered from the uteri of infertile camelids [41] [42]. Previously, E. coli was thought to be a non-specific pathogen associated with endometritis in mares and cows [43]. The isolation of E. coli and Staphylococcus spp from endometritis in this study were similar to findings of [44] who reported E. coli, Staphylococcus spp and Corynebacterium pyogenes associated with endometritis in ewes.
Normal E. coli strains can cause relevant diseases as the pathogenic ones by producing cytotoxic necrotizing factors, verotoxins and eae gene isolated from healthy cows, sheep and goats [45] [46]. Specific strains of E. coli (EnPEC) have recently been shown to be pathogenic for the endometrium, causing pelvic inflammatory disease (PID) in cattle [47].
The pattern of isolation of E. coli with other bacterial isolates in this study is similar to the isolation pattern of E. coli observed with Staphylococcus aureus, Bacillus and corynebacterium pyogenes from cases of endometritis in does, camels, ewes and bitches [18] [44] [48] [49] [50] [51].
[52], reported post partum infections are eliminated within 2 - 4 weeks of parturition and some of the uterine pathogens persist to cause subclinical endometritis [53] [54]. In this study, E. coli, Staphylococcus spp and Pseudomonas spp were isolated in association with pyometra, post-parturient metritis and post parturient emphysematous metritis. This was similarly reported in repeat breeders with mucopurulent discharges in cows [55] and camels [51].
From the present study, it was observed that E. coli, Staphylococcus spp, Proteus spp and Klebsiella spp were isolated in association with uterine caruncular atrophy, uterine congestion and melanosis. This could arise from spread of infections from other pathological sites where different lesions are occurring on individual genitalia. Uterine congestions occurred with endometritis while caruncular atrophy and melanosis were observed as single cases in this study.
There is a continuous clearance and recontamination of the uterine lumen postpartum for up to 7 weeks [9], pathological changes and inflammatory responses can be triggered by some bacteria which persist in the uterus and delay uterine involution [56], thereby lowering infertility. The severity of these inflammatory conditions is likely to be influenced by these types of bacteria colonizing the uterus [9].
The occurrence of ovulation prior to the expulsion of exudates and debris from the uterus and during the postpartum period has been shown to favour heavy growth of bacteria in the uterine environment. This leads to the retention of the corpus luteum (CL) and consequent impairment of the ability of the uterus to secret PGF2α [13] [57]. The number of bacteria colonizing the uterus and the level of uterine immune response may portend a risk factor for lowered reproductive efficiency because of increased inflammatory reactions and possible damages to the uterine tissues by direct action of the bacteria or its toxins [9] [11] [57] [58].
From the result of this study, the susceptibility pattern of E. coli to antimicrobials showed that Levofloxacin, Pefloxacin, Ciprofloxacin, Ofloxacin, Amoxyl and Gentamycin are most effective. Moderately active are Amoxycillin clavulanate, Streptomycin and Ceporex. The suceptibility of E. coli to Ampiclox was low. This finding agrees with previous reports of antimicrobial susceptibility of bacterial isolates from genitalia of goats, ewes and cattle [5] [35] [36] [55] [59] [60], but in variance with previous observations made by [61] in goats in southern Nigeria, where they reported that Gentamycin, Ofloxacin, Streptomycin, Ampicillin and Amoxycillin clavulanate were highly effective against E. coli.
Staphylococcus spp was highly susceptible to Levofloxacin, Amoxyl and Amoxycillin clavulanate. This finding contradicts previous reports in cattle and sheep [55] who found Ciprofloxacin as one of the most effective antimicrobial agent against Staphyloccocal uterine infections in dairy cows. Ciprofloxacin, Norfloxacin, Gentamycin, Ceporex and Rifampicin were moderately effective against Staphylococcus spp. However, Staphylococcus spp showed low susceptibility to Chloramphenicol and Erythromycin.
There is an increasing antimicrobial resistance in animals, which is complicating empirical selection of antimicrobial agents in veterinary practice [36]. These complications are continuously evolving in relation to factors such as the site of isolation, sex, age, species of the animal [62] and location where different antibiotic agents varied in their activity against bacterial isolates in Nigeria [36] [63].
The decrease in sample size in this study was as a result of damage incurred during the course of transport to the laboratory. The isolates were not identified at the specie level which is certainly a major limitation in this study. Also a reproductive history of live animals in correlation with clinical parameters would have elucidated on the importance of the isolates. Where possible, gynaecological evaluations in live animals should proceed with proper history, clinical and laboratory examination in order to determine specific aetiologies and generate useful data for effective diagnosis and management of genital disorders.
5. Conclusion
Bacteria colonizing the genital tract are similar in RS and WAD does. E. coli was the dominant bacteria isolated. E. coli and other isolates were found to be associated with genital disorders does. The bacterial isolates were found to be susceptible to antimicrobial agents tested. The potentials of these genital pathogens producing genital pathology and infection in goats are likely to be high but treatment could be achieved with the proper use of antimicrobials tested in this study. This could help in improving goat fertility, viable and productive small ruminant farm enterprise, increase in dietary protein and healthy wellbeing of the populace at large.
Acknowledgements
The assistance rendered by the people in sample collection and laboratory processing of the samples for this study is gratefully acknowledged. The authors are thankful to the Directors, Veterinary Teaching Hospital, FUAM and NVRI, Vom, for providing necessary facilities for this work.