Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 55 issue 8 (august 2021) : 946-950

Percutaneous Ultrasound Guided Cholecystocentesis and Microbiological Analysis of Bile in Chronic Diarrhoea of Cattle

Arati Hansda1,*, C.S. Randhawa1, M. Chandra2, J. Mohindroo3, S.S. Randhawa1
1Department of Veterinary Medicine, Ethics and Jurisprudence, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.
2Department of Veterinary Microbiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.
3Department of Veterinary Surgery and Radiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.
Cite article:- Hansda Arati, Randhawa C.S., Chandra M., Mohindroo J., Randhawa S.S. (2021). Percutaneous Ultrasound Guided Cholecystocentesis and Microbiological Analysis of Bile in Chronic Diarrhoea of Cattle . Indian Journal of Animal Research. 55(8): 946-950. doi: 10.18805/ijar.B-4195.
Background: Chronic diarrhoea is a frequently presenting complaint by the cattle owners and some presents as non-responsive to routine treatment. Such cases are often a diagnostic challenge because of wide ranging etiologies. In Indian context few data is available on prevalent causes of chronic diarrhoea in bovines. Salmonellosis is one of the important cause of chronic diarrhoea in crossbred cattle. Therefore, the study was conducted to investigate the role of Salmonella in chronic diarrhoea of crossbred cattle and to determine microbiological profile of bile in cattle with chronic idiopathic diarrhoea. 

Methods: Twenty (n=20) adult dairy cattle presented with the history of chronic diarrhoea at Large Animal Clinics, GADVASU, Ludhiana from September 2015 to June 2016 were selected for the study. Case histories were recorded, faecal, blood and bile samples were collected from 20 animals. Bile was collected through ultrasound guided cholecystocentesis (PUC) from 20 cattle and five healthy cattle. The faecal and bile samples were cultured for isolation and identification of bacterial pathogens using selective and non-selective media with special emphasis on isolation of Salmonella spp. Microscopic examination of faeces were done to rule out parasitic cause of diarrhoea and for acid-fast bacilli (AFB). The serum samples were employed to detect antibodies against Mycobacterium avium spp paratuberculosis using serum ELISA.

Result: Investigations have showed culture of bile and faecal did not yield Salmonella spp; however E.coli was isolated from 6 cows (30%), Klebsiella spp in 9 cows (45%) and Pseudomonas spp from 1 cow (5%) from bile culture of diarrhoeic cattle. PCR results of E.coli culture from bile showed hlyA virulence genes which expected band at 534 bp however these isolates were negative for other virulence gene of E.coli. It was observed in this study that bactibilia was associated with chronic diarrhoea in three fourth of the diarrhoeic cattle.
Chronic diarrhoea is one of the costly pathologic condition affecting livestock (Wyatt and Fayer, 2010) and often a diagnostic and therapeutic challenge in farm animals (Hassan, 2015).
A study in Indian dairy cattle and buffaloes has recorded the balantidiasis, amphistomiasis, salmonellosis, dietary errors, molybdenosis and johne’s disease as clinical reasons. However, the etiology was obscure in about 24 per cent of cases (Hassan, 2015). One of the possible causes in such cases may be chronic salmonellosis which could not be diagnosed because of failure to culture the organisms from faeces. Gall bladder wall, bile and mesenteric lymph nodes are the other sources rich in Salmonella for culturing (Woldemariuma et al., 2005; Arthur et al., 2008). In addition to that, microscopic examination of bile had also been demonstrated to improve diagnostic reliability of liver fluke infection over fecal examination in cattle (Braun and Gerber., 1992; Braun et al., 1995). In a study Fasciola hepatica eggs were detected in bile of 7 cows compared to 4 in fecal samples (Braun and Gerber., 1992) suggesting that bile was a better diagnostic sample for Fasciola hepatica as in the same study, the eggs of Dicrocelium dendriticum were detected in bile of 12 cows compared to none in fecal samples. Therefore, examination of bile appeared to be a reliable diagnostic tool for diagnosis of bacterial cholangitis or cholangio-hepatitis. Also microscopic examination of bile may use as an expedient means for the diagnosis of parasitic infections of biliary tract or of liver during prepatent or subclinical stage or for improving diagnostic sensitivity in cattle.
With the advent of ultrasonography, it is possible to collect bile in cattle. Percutaneous ultrasound-guided cholecystocentesis (PUC) is a minimally invasive technique that permits the collection of a large bile sample for standard diagnostic analysis (Bacterial culture) suspected with cholangiohepatitis (Karine et al., 2003). It was, therefore, hypothesized, that the bile might be better sample in chronic diarrhoea cases, where faecal culture were negative and hence this investigation was conducted.
The experiment was carried out at large animal clinics of Guru Angad Dev Veterinary and Animal Science University, Ludhiana from September 2015 to June 2016.
Selection of animals
Twenty (n=20) adult dairy cattle presented with the history chronic diarrhoea were selected for this study. Disease history was recorded with respect to appetite status, effect on body weight, feeding regime, other clinical signs associated with the onset of diarrhoea, number of animals affected at outset, number of animals affected during last five years in the herd, previous treatment undertaken and the response. Each cow was subjected to general physical and rectal examination for any intestinal or other intra-abdominal abnormalities (masses or lymphadenopathy). Twenty five cattle (twenty diarrheic cattle and five healthy cattle) with no other clinical abnormality, negative for faecal parasitic infection as well as for serum antibodies against Mycobacterium avium var. paratuberculosis were selected for this study.
Blood sample
Blood was collected by jugular venepuncture for serum harvesting.
Faecal sample
Fecal samples were collected directly from rectum.
Bile Collection method
The skin over 10th-11th intercostal space on the right side of the abdominal wall was shaved and local anaesthetic solution (2% lignocaine) was infiltrated subcutaneously at the level of 2/3rd of the dorsum of 10th and 11th intercostal spaces on right side (Fig 1). Initially, gall bladder was located by 2.0 to 5.0 MHz convex transducer (Fig 2). Later, first quadrate lobe of liver was located and transducer was manipulated by sliding and rotating to visualise gall bladder as echo-lucent area. The site was then cleaned with ethyl alcohol and with 10 ml 23 gauge sterile spinal needle with stylet was inserted to aspirate bile.

Fig 1: Ultrasonographic image of gall bladder at 10th ICS.


Fig 2: Ultrasonography procedure to locate gall bladder in standing animal.

Processing of samples
Serum samples were tested for antibodies against Mycobacterium avium var.paratuberculosis by serum ELISA (IDEXX Laboratories, IDEXX production Animal Services).
Faecal samples were examined for parasitic infection by sedimentation and floatation method for detection of parasitic cysts or ova. Also, faecal samples were subjected to isolation of bacteria with emphasis on isolation of Salmonella spp. Faecal sample was subjected to Ziehl -neelsen Staining for detection of faecal shedding score of acid-fast bacilli (AFB).
Isolation and identification of Salmonella and other bacteria from bile
Approximately, 1 ml of bile sample was directly inoculated in 100 ml of buffered peptone water (BPW) and incubated overnight at 42°C and then 0.1 ml of it was transferred to 10 ml of Rappaport Vassiliadis (RV) broth, and incubated overnight at 42°C. From selective enrichment, samples were inoculated on Hektoen enteric agar (HE) and Brilliant Green Agar (BGA) and incubated overnight at 37°C. The suspected colonies were subjected to biochemical identification.
For, E. coli, Klebsiella and Pseudomonas, from BPW one loop full was streaked on Eosin Methylene Blue Agar plates and nutrient agar respectively for overnight at 37°C. The suspected colonies were subjected to biochemical identification.
Polymerase chain reaction
PCR was performed for identification of Salmonella from faeces and virulence gene of E.coli from bile of cattle as per the standard technique.
Selection of animals
Consistency of faeces was watery in 7 cows (7/20,35%) and poorly formed in 13 cows (13/20,65%). Recurrent diarrhoea was recorded in 14 cows(14/20,70%) and persistent in 6 cows (6/20,30%). A notable observation from the history suggested that diarrhoea was not an endemic in nature, as no other animals developed diarrhoea during last 5 years.
Faecal examination
Faecal parasitic examination was unremarkable in more than half of the cases (13/20, 65%). Subclinical Balanditium coli (occasional cyst) infection was detected in 5 cows (5/20, 25%), occasional fasciola and strongyle eggs were detected in cow each.
Faecal Acid-fast bacilli (AFB) load and serum Mycobacterium avium paratuberculosis (MAP) antibodies status
One of two diarrhoeic cows with heavy AFB load (4+) showed positive for serum MAP antibodies in ELISA and other cow was suspected MAP antibodies. Out of 3 cows with high AFB load (3+) showed suspected for MAP antibodies in one cow and negative in 2 cows, 4 cows with moderate AFB load (2+) were suspected in 2 cows and negative for 2 cows and 3 cows with low AFB load (+) were negative for MAP antibodies. No MAP antibodies were detected in cow with negative for AFB load (Table 1).

Table 1: Faecal AFB load and serum MAP antibodies status.

One cow which was positive in ELISA for MAP antibodies, after treatment it showed complete clinical recovery in terms of faecal consistency, body weight and milk yield. So in this study we can hypothesized that it was not the case of Johne’s disease. Clark et al., (2008) reported ELISA had moderate sensitivity (30 to 50%) and 100% specific.
 Collection of bile
Collection of bile was attempted in twenty five diarrhoeic animals and it was successful in twenty cows. Gall bladder was markedly distended in twelve and moderately distended in eight diarrhoeic animals. Gall bladder was markedly collapsed and reduced in size in five animals where attempt was unsuccessful (Fig 3). All the cows were observed for two weeks for general attitude, effect on appetite and any evidence of illness. None of the sampled cows developed any complication.

Fig 3: Ultrasonographic image of reduced size Gall Bladder.

The gall bladder was visible at 10th intercostal space in 14 cows and at 11th intercostal space in 6 cows. It was consistently observed at 10th intercostals space in all healthy control cows. Successful percutaneous cholecystocentesis procedure was performed in 30 cows on right side of 9th, 10th and 11th intercostals spaces (Braun and Gerber, 1992).
In a comparative ultrasonographic study of normal bovine spleen and liver, the location of gall bladder was from the 9th-12th right intercostal spaces (Imran et al., 2011). The location of the gallbladder varied from the 12th to 9th intercostals space. In this study gall bladder of cattle was imaged as a tear drop or a pear shaped anechoic structure with hyperechogenic wall near to quadrate lobe of liver. No morphological difference in gallbladder wall or its contents was appreciable in animals with positive bacterial culture. The bile was light green with thinner consistency in twenty diarrhoeic and viscous dark green in non-diarrhoeic cows. The pH varied from 7.5-8.5 in both groups. The collection of bile by ultrasound guided cholecystocentesis in cattle had been described infrequently in cattle (Braun and Gerber, 1992; Braun et al., 1995) though, the gall bladder, its contents and its changes in wall had been described during abdominal ultrasonography in cattle (Tharwat, 2012; Braun et al., 2013). Therefore, it may be extrapolated from this study that sample of bile can be safely collected and used for ante mortem diagnosis of biliary tract diseases. Previously, E. coli, Bacteroides spp, E.coli and Trueperella pyogenes had been cultured from liver biopsies in cattle diagnosed for cholangiohepatitis (Gomez et al., 2017). Studies in dogs had reported that bile culture was advantageous as it yielded higher positive culture rates than hepatic tissue (36% vs 16%) and positive bile culture increased by 64 per cent in chloecystitis (Wagner et al., 2007).
The association between ultrasonographic findings of gall bladder and bile culture had shown a sensitivity, specificity and accuracy of 82, 55.7 and 61.5 per cent in dogs and cats (Schiborra et al., 2017).
Bacterial micro biota of bile
All the samples of bile were negative for Salmonella spp. Previously, Salmonella spp. had been isolated from bile collected from slaughtered cattle, although, isolation was less frequent from bile compared to gall bladder epithelium (Woldemariama et al., 2005). In other study, Salmonella organisms were isolated from the bile as well as from the gall bladder epithelium, but in larger quantities from the gall bladder epithelium (Dias et al., 2010).
Interestingly, E. coli, Klebsiella spp and Pseudomonas spp. were isolated from bile of 16 out of 20 diarrhoeic cattle; E. coli from 6 (30%), Klebsiella spp in 9 cows (45%) and Pseudomonas spp in 1 cow (5%). Sample of bile was sterile in remaining 4 diarrheic (25%) and all the healthy control cows. A previous study reported E.coli O157:H7 from bile of only 2.7 per cent of the healthy slaughtered cattle and suggested that gall bladder was not a common site of E. coli O157:H7(Reinstein et al., 2007). It was reported that localization of E. coli was higher in immune-suppressed animals than the healthy cows (Dean-Nystrom et al., 2008). From the bacterial profile of bile, the most likely route of infection in these diarrhoeic cows appeared to be the ascending of enteric pathogens into biliary tract from intestine. Similar to the present study enteric pathogens (E. coli, Enterococcus spp. Streptococci spp.) were the most common causes of bacterial cholangitis or cholangio-hepatits in dogs, cats and also in man (Wagner et al., 2007; Policelli-Smith et al., 2017).
Examination of E. coli isolates for virulence by PCR showed hlyA virulent genes, in which amplified PCR product showed expected bands at 534bp (Paton and Paton, 1988) (Fig 4). These isolates, however, were negative for other virulence genes viz. stx1, stx2 and eaeA. This finding probably lent support to the fact that these organisms were not simple contaminants and might be pathogenic at the infected tissues (biliary tract, intestine).

Fig 4: Ethidium bromide-stained 1.5% agarose gel showing results of electrophoresis of products of the PCR reaction. A 534- bp band is seen in 6 lane with the product of the PCR for hlyA virulence gene of E. coli.

The important question was whether these organisms can be considered to be involved in initiation of chronic enteritis in cattle. In our opinion the role of biliary infection in diarrhoeic cows cannot be denied because of (a) isolation of pure culture of E. coli, Klebsiella spp and Pseudomonas spp. bacteria from bile and presence of virulence gene in E. coli; (b) fifteen of 20 diarrheic cows showed bacterial growth in bile compared to none in control group. (c) diverse community of intestinal bacteria exist in a delicate balance and were prone to disruption by dietary changes or antibiotics or infection with pathogenic bacteria to cause dysbiosis (Koboziev et al., 2014). The dysbiosis could result intestinal dysfunction including inflammation of intestinal mucosa.
Antibiogram of bacterial isolates from bile
All the isolates of E.coli were sensitive to enrofloxacin, ampicillin, amoxicillin, co-trimoxazole, cefuroxime, cefoperazone and sulphadiazine. Sensitivity was moderate (66.6%) for sterptomycin, ceftriaxone and was lowest (33.3%) for tetracycline, nalidixic acid and resistant to penicillin. Klebsiella showed highest sensitivity for enrofloxacin (100%), followed by gentamicin (87.5%), ampicillin (87.5%), cefuroxime (87.5%) and cefoperazone (87.5%). Sensitivity was moderate for streptomycin (75%), amoxicillin (75%), sulphadiazine (75%), co-trimoxazole (62.5%) and ceftriaxone (62.5%). Sensitivity was lowest for tetracycline (50%), nalidixic acid (50%) and completely resistant to penicillin.
Earlier report recorded that bile origin E. coli strains were resistant to streptomycin, sulphamethoxazole and tetracycline compared to moderate sensitivity to streptomycin and tetracycline (Byme et al., 2003). In another study it was reported that bile origin E.coli were resistant to sulphamethoxazole, cephalothin and ampicillin which was in contrast to the present study (Schroeder et al., 2002).
Klebsiella spp. showed highest sensitivity for enrofloxacin (100%), followed by gentamicin (87.5%), ampicillin (87.5%), cefuroxime (87.5%) and cefoperazone (87.5%). Sensitivity was moderate for streptomycin (75%), amoxicillin (75%), sulphadiazine (75%), co-trimoxazole (62.5%) and ceftriaxone (62.5%). Sensitivity was lowest for tetracycline (50%), nalidixic acid (50%) and completely resistant to penicillin. One isolate of Pseudomonas spp. isolated from bile showed sensitivity to enrofloxacin, ceftriaxone, ampicillin, amoxicillin, cefuroxime, ceftriaxone, nalidixic acid, penicillin, gentamicin, streptomycin, tetracycline. There was complete resistance to co-trimoxazole and sulphadiazine.
Therefore, all the three bacteria were highly sensitive to enrofloxacin. Four of six diarrheic cows showing bactibila that indicates microbial flora in bile and treated with enrofloxacin recovered completely with 5 days of treatment. This observation also adds to the hypothesis that infected bile was likely to be the triggering factor or cause of chronic diarrhoea, although a larger data is required to prove or contradict this hypothesis.
Chronic diarrhea is a symptom complex with wide range of aetiologies. Chronic Salmonellosis is one of the important cause. There are many published data of faecal isolation of these bacteria but very few data on bile isolation. So it was hypothesized that bile may be better source for isolation of bacteria as enteric bacteria including Salmonella are resistant to the effect of bile and play role in the development of carrier state. In this study we can found that culturing of bile for isolation of Salmonella does not offer any advantage over faecal culture. Although other bacteria like E.coli, Klebsiella and Pseudomonas spp are present in bile of cattle manifesting chronic diarrhea.
All authors acknowledge the support of the Dean, PG Faculty of Veterinary and Animal Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab for carry out the experiment.

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