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).
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.
The gall bladder was visible at 10
th intercostal space in 14 cows and at 11
th intercostal space in 6 cows. It was consistently observed at 10
th intercostals space in all healthy control cows. Successful percutaneous cholecystocentesis procedure was performed in 30 cows on right side of 9
th, 10
th and 11
th 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 9
th-12
th right intercostal spaces
(Imran et al., 2011). The location of the gallbladder varied from the 12
th to 9
th 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).
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.
