Clinicopathologic findings associated with urinary tract problems in working donkeys, Southern Ethiopia: a case study Berhanu Mekibib, Edget Abrham, Asmamaw Kassaye, Dawit Tesfaye & Kebede Amenu Comparative Clinical Pathology ISSN 1618-5641 Volume 24 Number 2 Comp Clin Pathol (2015) 24:417-422 DOI 10.1007/s00580-014-1918-6
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Author's personal copy Comp Clin Pathol (2015) 24:417–422 DOI 10.1007/s00580-014-1918-6
ORIGINAL ARTICLE
Clinicopathologic findings associated with urinary tract problems in working donkeys, Southern Ethiopia: a case study Berhanu Mekibib & Edget Abrham & Asmamaw Kassaye & Dawit Tesfaye & Kebede Amenu
Received: 10 August 2013 / Accepted: 13 March 2014 / Published online: 1 April 2014 # Springer-Verlag London 2014
Abstract A study was conducted in Southern Ethiopia to determine the extent and characteristics of urinary tract problems and the associated clinicopathological findings in donkeys. The study was carried out by sampling a total of 1,441 donkeys from Hawassa, Leku, and Yirgalem towns of Southern Ethiopia from October 2008 to March 2009. Out of 52 suspected to have urinary tract problems based on owners’ complaints and physical clinical examination, urine samples were aseptically collected from 23 of the cases by catheterization for physical, microscopic, and bacteriological analyses. The common urinary tract problems based on history and physical clinical examination were prolonged protrusion of penis with dribbling of urine after urination 28 (53.8 %), painful urination 18 (34.6 %), and frequent changes in posture during urination 15 (28.8 %). Management-related problems like injury to the external genitalia were also noted in 11.5 % of the donkeys during physical clinical examination. Laboratory investigation of urine samples from 23 donkeys presented for urinary problems showed that 18 (78.3 %) had bile pigment, 5 (21.7 %) protein, and 7 (30.4 %) altered pH. The microscopic examination of the urine samples revealed the presence of crystals, pyuria, and microhematuria. Upon bacteriological culture, different species of bacteria were isolated from the urine samples which include Streptococcus, Escherichia coli, Corynebacterium, Staphylococcus, Enterobacter, Pseudomonas, and Proteus. Overall, result of the present study showed that urinary tract problems are low in prevalence but still with significant health implications due B. Mekibib : E. Abrham : D. Tesfaye (*) : K. Amenu School of Veterinary Medicine, Hawassa University, P.O. Box 5, Hawassa, Ethiopia e-mail:
[email protected] A. Kassaye Donkey Sanctuary Ethiopia, Hawassa Site Project, Hawassa, Ethiopia
to donkeys are not given proper care with regard to watering and feeding practices. Keywords Urinary tract problems . Donkeys . Urinalysis . Pyuria . Bacteriuria . Ethiopia
Introduction Equines are kept in different agroclimatic zones of Ethiopia playing various socioeconomic roles (Agajie et al. 2000; Dinka et al. 2006). According to FAOSTAT (2011), the equine population of the country is estimated at 5.7 million donkeys, 2.03 million horses, and 0.38 million mules. Equines are used as pack animals in different parts of the country and they carry various goods such as water, fire woods, agricultural products, and construction materials (Howe and Garba 1997). Equines are also used for transporting people in the form of pulling cart or on the back of the animals. Underdeveloped transportation infrastructure in much part of the country make equines the major means of transportation for both goods and people (Kidanmariam 1997). Though equines are contributing a very crucial role with regard to transportation of goods and humans in the country, the welfare of these animals is generally very poor associated with inadequate healthcare and management conditions (Biffa and Woldemeskel 2006). There are also a number of diseases that affect equines, thereby reducing the power they can provide as pack or transport animal (Curran et al. 2005). According to Tiselius et al. (2000), abnormalities of the urinary tracts can be originated from damage on the kidney, ureters, bladder, or urethra as a result of exposure to chemical, infectious, or physical agents. Extensive studies have been conducted on various health aspects of donkeys in Ethiopia by various scholars including Feseha (1998), Getachew (1999), Yoseph et al. (2001), Yoseph et al. (2005), and Ayele et al.
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(2006), mostly focusing on parasitic diseases. On the other hand, information on the urinary tract problems in working donkeys is lacking in the country. Therefore, the present study was carried out to assess the extent of urinary tract problems of working donkeys and the associated clinicopathologic findings in the urine of these animals.
Materials and methods
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Urine sample collection and processing Urine samples were aseptically collected in sterile universal bottles by catheterization. The catheters were washed and autoclaved after each sample collection and were reused. The collected urine samples were properly labeled, placed in ice box and transported to Veterinary Pathology laboratory of Hawassa University and examined within 2 to 3 h of collection. Urinalysis was used to assess the functional capacity of the urinary tract and to reveal the presence of abnormal cells, pigments, or bacteria in the urine (Van Steenhouse 2002).
Study area Physical and chemical examination The study was carried out in three selected veterinary outreach sites of Donkey Sanctuary Ethiopia (i.e., Hawassa, Leku, and Yirgalem towns). Donkey Sanctuary is a United Kingdom (UK)-based international nongovernmental organization working on equine health and welfare in various developing countries of the world including Ethiopia. The Donkey Sanctuary Ethiopia offers free veterinary clinical services to donkeys and educates donkey owners on animal welfare and management. The three towns, namely, Hawassa, Leku, and Yirgalem are located 270 km, 295 km, and 310 km, respectively, south of Addis Ababa. Donkeys in the areas pull carts and transport goods or people because of, apparently, motorized transport systems are not affordable or in some places are inaccessible due to undeveloped road infrastructure. Donkeys either by pulling cart or as pack animal transport various goods such as construction materials (building block, sand, gravel, and stone), agricultural products and by-products (from field to homes and then to market), firewood, and drinking water in the area. Specifically, cart-pulling donkeys are the main livelihood and income-generating activities for the owners of the animals. The feeding system of donkeys involves supplemental feeding with wheat bran, chopped sugar cane, and household food waste during working time and grazing on communally lands as well as roadsides.
Case identification and sampling procedure Donkeys included in the present study were identified from consecutive cases of donkeys presented to the three outreach sites of the Donkey Sanctuary Ethiopia described above during October 2008 to March 2009. Out of 1,441 donkeys presented to the outreach veterinary clinics for various reasons, 52 animals were suspected to have urinary tract problems based on owners’ complaints and physical clinical examination. Of those donkeys with complaints of urinary tract problems, urine samples were aseptically collected by catheterization for further physical, microscopic and bacteriological analyses from 23 of the cases. Owners could not cooperate for the remaining cases for urine sample collection.
Color, odor, foaminess, turbidity, and specific gravity of the collected urine sample were evaluated immediately after collection. The urine was observed in a test tube for its color and turbidity. The color was recorded from colorless to any known color, whereas the turbidity was recorded as clear, flocculent or cloudy depending on the suspended dry matter (Coles 1986). For the presence of protein, the amount and rate of disappearance of foam was appreciated after shaking the urine in a test tube. The color of the foam was simultaneously checked for the presence of bile and bile pigment. The specific gravity was estimated by using pocket refractometer (Latimer 2011). Analysis of the chemical properties of urine was carried out on the fresh and aseptically collected urine sample. Hydrogen ion concentration (pH) and the presence of protein and bile pigment were determined according to routine procedures described by Coles (1986). Microscopic and bacteriological examination About 5 ml thoroughly mixed urine sample was taken in a graduated conical tube and centrifuged for 3.5 min at 1500 revolution per minute (rpm). The supernatant fluid was poured-off leaving the sediment. The tube was gently shaken to breakup sediments. Then one drop was placed on a clean glass slide and covered with a clean cover slip and examined unstained under the microscope with subdued light. Similarly, smears were prepared from the sediment, air dried, and stained with modified Giemsa (MayGrünwald-Giemsa, Merck KGaA, Darmstadt, Germany) for further investigation on the cellular response of the host and infectious agents. The stained smears were then thoroughly examined under microscope (×10, ×40 and oil immersion objectives). Part of the aseptically collected urine of each sample was inoculated on blood agar and MacConkey agar and incubated overnight to get primary culture of bacteria. Presumptive identification of the bacteria on primary culture was made
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on the basis of colony morphology, hemolytic characteristics, Gram stain reaction (shape and arrangements of the bacteria) and catalase test. Gram negative bacteria grown on MacConkey agar were tentatively identified based on growth characteristics on MacConkey agar, oxidase reaction, catalase test, growth on triple sugar iron (TSI) agar, and on “IMViC” (indole, methyl red, Voges-Proskaur, and citrate) test (Quinn et al. 2002).
Results Based on owners’ complaint, out of the total 1,441 donkeys presented to the three working sites of Donkey Sanctuary Ethiopia outreach sites, 52 cases (3.6 %) were tentatively diagnosed as having urinary tract problems. This was further supported by the presence of distended bladder in 28 (53.8 %) of the donkeys during rectal examination. Managementrelated problems like injury to the external genitalia were also noted in six donkeys (11.5 %) during physical examination (Table 1).
Table 2 Summary on the physical and chemical evaluation of urine samples collected from donkeys (n=23)
Physical examination Color Watery Yellowish/amber Deep brown Red Turbidity Clear Cloudy Very cloudy Odor Normal Fetid Specific gravity Normal High Chemical examination Protein Negative Positive Bile pigment Negative Positive pH 8.5
Number of cases
Percent
3 17 2
13.0 74.0 8.7
1 7 14 2 20 3 21 2
4.3 30.4 60.9 8.7 86.9 13.1 91.3 8.7
18 5 18 5 1 16 6
78.3 21.7 78.3 21.7 4.3 69.6 26.1
Physical and chemical evaluation of urine Physical and chemical evaluation of the urine samples collected from 23 donkeys with signs of urinary tract problems revealed 2 deep brown urine samples (8.7 %), 1 bright red urine (4.3 %), 2 with high specific gravity and 5 rich in protein (21.7 %) (Table 2).
Table 1 Summary of owners’ complaints and physical clinical examination results in donkeys with urinary tract problems (n=52) Signs/symptoms
Frequency
Dribbling of urine Distended bladder Weight loss Painful urination Frequent posturing of urination Colic Fever Loss of appetite Fecal impaction Congested mucus membrane Wound on the scrotum and prepuce Grunting while walking and upon palpation Swollen and hot testis Penile discharge Red urine
28 28 26 18 15 13 12 11 8 6 6 3 3 3 2
Normal range of specific gravity was taken at 1.008–1.040 and pH from 7.5 to 8.5 (Dowling et al. 2000)
Microscopic and bacteriological examination of urine sediments Examination of urine sediment, as a routine part of a complete urinalysis, revealed microburia particularly bacteria, cells, casts, and crystals (Table 3). The results of bacteriological analysis showed that out of 23 aseptically collected urine samples, a total of 30 bacterial isolates were identified. Pure colony was recovered from 8 urine samples whereas 11 of the urine samples showed mixed colonies and no bacterial isolate was detected in 4 of the urine samples. Streptococcus spp., E. coli, and Corynebacterium spp. were the most commonly isolated bacteria with frequency of 13, 6, and 3, respectively. Moreover, other bacteria including Staphylococcus spp., Enterobacter spp., Pseudomonas spp., and Proteus spp were also isolated (Fig 1).
Discussions Urinary tract problems, urinary tract infections (UTIs) in particular, reportedly account for between 5 and 10 % of canine and 0–1 to 1 % of feline veterinary visits (Dunning and Stonehewer 2002). However, it is rare in other domestic animals including donkeys (Chandler 2002).
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Table 3 Summary of urine sediment examination results in donkeys with signs of urinary tract problems (n=23) Kinds of urine sediments
Number of cases
Percent
Crystals
Uric acid Amorphous urate Calcium carbonate Calcium oxalate
2 2 23 3
8.7 8.7 100.0 13.0
Cells
Neutrophils (pus cells) Intact red blood cells Red blood cells (ghost) Transitional epithelial cells Hyaline Waxy Mucus threads resembling casts Rod Cocci
9 5 3 13 3 6 9 2 5
39.1 21.7 13.0 56.5 13.0 26.1 39.1 8.7 21.7
Casts
Bacteria
Distended bladder, dribbling of urine, weight loss, painful urination, frequent posturing of urination, and colic were the most common complaints raised by the owners. According to Kobluk (1995), abnormal urination and weight loss are the most common presenting complaints in horses with urinary tract disease. The study revealed that 3.6 % of donkeys in the study areas were having different sorts of urinary tract problems. A postmortem study conducted in donkeys in Egypt by Bakeer et al. (2008) showed an overall prevalence of acute cystitis (19 %), acute ureteritis (14 %) subacute tubulointerstitial nephritis (50 %), renal fibrosis with cysts (13 %), membranous glomerulonephritis (3 %) proliferative glomerulonephritis (6 %), glomerulosclerosis (3 %), hemorrhage (6 %), and nephrolithiasis with chronic pyelonephritis (3 %). The relative lower prevalence of urinary tract problems in the current study could be due to the variations in the diagnostic methodologies. In the our case, clinical examination combined with urinalysis and bacteriological diagnosis was applied which in turn could underestimate the problem as the sensitivity of this
Pseudomonas 6.7%
Proteus 6.7%
Enterobacter 6.7%
Streptococcus 43.3%
Staphylococus 6.7%
Corynebacterium 10.0%
E.coli 20.0%
Fig. 1 Summary on the common bacteria isolated from aseptically collected urine samples (n=23)
methodology is generally lower as compared to postmortem examination. Out of the 23 urine samples, 6 urine samples (26.1 %) were with alkaline pH and 1 sample (4.3 %) was with acidic pH while the remaining 16 (69.5 %) were in the normal range. The urine pH of healthy animals could be influenced by diet, exercise, infection, or any factor that delays the frequency of urine clearance. According to Latimer (2011), causes of urine retention yield higher urine pH because of bacterial catabolism of urea to ammonia. The higher urine pH found in the present investigation of may be due to the retention of urine associated with urinary tract problems including lower urinary tract infection. Bile pigment test indicated that 5 (21.7 %) of the donkeys were positive. Brar et al. (2000) stated that unconjugated bilirubin, being bound to albumin, cannot pass an intact glomerular membrane. Latimer (2011) also stated that the presence of bilirubin indicates obstruction to bile flow and regurgitation of conjugated bilirubin into the blood; it is mostly seen in hemolysis, liver disease, extra hepatic biliary obstruction and starvation. Moreover, unconjugated bilirubin can be noticed in the urine when the renal tubules are damaged (Parrah et al. 2013). Proteinuria was detected in 5 (21.7 %) of the urine samples. According to Latimer (2011), this condition is usually associated with hemorrhage and inflammation within the urinary tract. Occult blood cells, myoglobin, leukocytes, presence of bacteria, glomerular and/or tubular diseases, and deposition of castes elevate urine protein level accompanied by certain other extra hepatic factors like fever and cardiac problems (Parrah et al. 2013). The results of nine urine samples (39.1 %) indicated high counts of mucus casts. Equine urine is very foamy and cloudy when it is first passed and it disappears after some time. This is due to the large amount of mucus in fresh equine urine (Thomas 2008). However, the presence of mucus cast usually indicates the presence of chronic insult on the lower urinary tract especially on the renal pelvis. In this study, urine sediment examination revealed calcium carbonate deposition in all 23 samples examined (100 %). Calcium carbonate crystals are the most common crystals in equine urine and are considered a normal finding (Dowling et al. 2000). In their endoscopic study on 68 horses, Laverty et al. (2008) reported that equine uroliths are most often composed of calcium carbonate and are most commonly found in the bladder (60 %) or urethra (24 %). The pathogenesis of urinary calculi is uncertain, but it is likely that some underlying metabolic or inflammatory process precedes their development. Calculi may develop when urine stasis or tissue damage provides a nidus up on which minerals may be deposited or crystals may aggregate (Coles 1986). Calcium oxalate was found in three of the urine samples (13.0 %). This crystal is seen in animals poisoned with ethylene glycol used as antifreeze in vehicles and certain plant
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toxicities (Brar et al. 2000). From the animals behavior point of view and low availability of ethylene glycol in the study area, the presence of this crystal in urine could indicate the donkeys most probably consumed plants rich in oxalate (Coles 1986). Among the urine samples, nine (39.1 %) had neutrophils in their urine sediment. Increased number of WBCs (pus cells) in the urine sediment, which is termed as pyuria, is the consequence of urinary tract infection (Brar et al. 2000). Dowling et al. (2000) describes elevation in urinary leukocytes as indicative of inflammation in the urinary tract and when combined with bacteria are reflective of infection. In this study, the pyuria accompanied by bacteriuria as rod-shaped bacteria were found in 2(8.7 %) of the urine samples and Cocci in 5 (21.7 %) of the samples. As Brar et al. (2000) indicated, increased number of bacteriuria suggests cystitis or pyelonephritis which is derived from urothelium from renal pelvis to urethra. Transitional epithelial cells were found in one case (4.34 %). Presence of transitional epithelium in urine is indicative of cystitis or pyelonephritis (Brar et al. 2000), because the integrity of the urothelium will be lost and hence cells will slough. During physical examination of urine, macrohematuria was observed in two (8.7 %) of the samples. In addition to this, microhematuria was noted in 8 (34.8 %) samples during microscopic examination of urine sediment. According to Schott and Hodgson (2000), increased urine flow during exercise might have contributed to the infrequent observation of gross hematuria in these working donkeys. The hematuria could be intrinsic in origin resulting from abnormalities such as uroliths and cystitis; or it can also be due to gross damage to the external genitalia because of beating by owners or friction with harness. In this regard, authors had a chance to see some owners beating their donkey at the inguinal region so as to respond and move fast. Hyaline casts were observed in three samples (13 %) using dim light. These were clear colorless and highly retractile. As these castes dissolve in alkaline urine of herbivores, presence of hyaline casts suggests mild renal irritation commonly seen in fever, exercise or general anesthesia. The waxy casts observed in the current study (n=6, 2.1 %) may indicate chronic degeneration of kidney. Bacteriological culture of the 23 samples showed no bacterial growth in 4 (17.4 %) samples, 8 (34.8 %) pure colonies, and 11 (47.8 %) mixed colonies. In the bacteriological analysis of the samples, Streptococcus spp. shows 42 % of the bacterial isolate followed by E. coli (20 %) and Corynebacterium (10 %); whereas each of Staphylococcus, Enterobacter, Pseudomonas, and Proteus isolates accounted to 7 %. This finding matches with the report of Dowling et al. (2000) which implicates E. coli, Proteus mirabilis, Enterobacter spp., Staphylococcus spp., Pseudomonas spp., Streptococcus spp., and Corynebacterium spp. as the most
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common causes of urinary tract infection in equines. As observed in this study, if freshly passed urine has a significantly alkaline pH (8 to 8.5) and an ammoniacal odor, it suggests infection with a urease-producing organism, staphylococci or Proteus spp. (Bush 1992). In conclusion, the present study showed that urinary tract problem was found to be low in donkeys presented for various problems. However, samples collected from nine cases (39.13 %) were showing pyuria, bacteruria, and signs of stranguria, the cases which most probably be due to infectious in origin, particularly on the lower urinary tracts. Moreover, it is recommended that postmortem be conducted on such cases to further identify the exact nature of the disorder. Being the first in its kind in Ethiopia, the present study can serve as a preliminary finding and further detail investigation is recommended on the various aspects of urinary tract problems in donkeys.
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