Ancillary Diagnostics for Gastrointestinal Disease
ACVIM 2008
Meredyth L. Jones, DVM, MS, DACVIM
Manhattan, KS, USA

Rumen Fluid Examination

Rumen fluid examination is a diagnostic aid for animals exhibiting abdominal distension, reduced fecal output, anorexia, and abnormal rumen texture or motility. It is also useful for screening cattle herds for Subacute Ruminal Acidosis (SARA).

Rumen fluid may be obtained by passage of an orogastric tube with aspiration by a dosing syringe or reversed stomach pump. The use of a weighted tube allows passage of the tube through the fiber mat ventrally into the fluid portion of the rumen contents. This method may result in salivary contamination, which significantly affects some analytes, including pH, Na, K and methylene blue reduction time.

Rumenocentesis completely avoids salivary contamination and is therefore the preferred method for SARA screening. It usually results in a smaller sample volume and carries a low risk of subcutaneous abscessation and localized peritonitis.1 It should also be avoided in cows with LDA and in late gestation. To obtain fluid, a line should be drawn horizontally extending cranially from the stifle, ending about 8" caudal to the last rib in adult cattle. This endpoint should be clipped and disinfected and a tail jack applied. A 14-16ga, 2-5" needle should then be thrust directly into the rumen and 12-20mL of fluid withdrawn into a syringe.

Once collected, the fluid should be placed in a clean vial and either analyzed immediately or kept at body temperature until timely analysis can be performed.

Table 1. Interpretation of rumen fluid values in cattle.

 

Normal

Abnormal

Color

Corn silage/straw: yellow brown

Black green: putrefaction

Concentrates: olive brown

Milky grey: lactic acidosis

Pasture: green

 

Odor

Aromatic

Acidic, sour: lactic acidosis

Consistency

Slightly viscous

Rotting: putrefaction

 

Ammonia smell: urea toxicity

pH

Grass: 6-7

5.5-6.0: pastured cattle with early lactic acidosis;
rumen drinkers

Grain: 5-6

<5.5: lactic acidosis, except well-adapted
feedlot cattle

 

7-8: anorexia for >2 days; simple indigestion

 

>8.0: saliva contamination, putrefaction,
urea toxicity, spoiled feed,

 

manure contamination of feed, coliforms,
MgOH therapy2

Microscopy

>40 protozoa/lpf (10x)

Large protozoa die first from acidosis

3 sizes, rapid movement

 

Gram's Stain

G neg predominate

upwards arrowG pos: acidosis

Met. Blue Red. Time

2-6 min

>10-15 min: inadequate anaerobic bacteria

Concentrates: 2-4 min

 

Forage only: 5-6 min

 

Sed. Time

4-8 min

<4-8 min to sedimentation: inactive fermentation

Rumen [Cl-]

<30mEq/L

>50 mEq/L: abomasal reflux, salt therapy or intake

Rumen fluid pH is best measured using a pH meter3, which is less affected than pH paper by the color of the rumen fluid. A drop of fluid may be placed on a warm glass slide and examined using a microscope. On low power, numerous protozoa should be seen moving rapidly across the slide. Three general sizes of protozoa should also be identified. The large protozoa are the most sensitive to lactic acidosis and will be the first to be lost in cases of rumen acidosis. This slide may then be dried and Gram stained to assess the ratio of G pos and G neg bacteria.

For herd-level SARA testing, sampling should only occur in light of clinical syndromes. Twelve cows on each feed should be sampled, with cows 3 weeks into lactation and cows 45-150 days into lactation considered. Sampling in component-fed herds should occur 2-4h after feeding, while herds consuming TMR should be sampled 6-8h post-feeding. If >25% of samples animals have a rumen pH <5.5, the herd is at risk for experiencing syndromes associated with SARA. An individual result >5.8 is considered negative for SARA. As with all population testing, if the results fall very near to the cutoff for risk, additional samples should be obtained and analyzed.4

Methylene blue reduction time is determined by mixing 10mL of fresh rumen fluid and 0.5mL of 0.03% methylene blue into a clear tube. The time taken for the rumen fluid to be decolorized back to its original color is then determined, which is the result of normal anaerobes reducing the methylene blue. Sedimentation time is determined by placing rumen fluid into a clear tube and watching for feed particles to sink to the bottom of the tube. With active fermentation, gas bubbles cause the feed to float. Samples that sediment quickly have inadequate fermentation.

Rumen chloride concentration is a useful test that may be submitted to some labs. Abomasal HCl reflux into the rumen occurs due to vagal indigestion, small intestinal obstruction or ileus. Because of highly effective rumen buffering systems and volume, this HCl will not cause the rumen pH to change, but will alter [Cl-]. Rumen [Cl-] should be interpreted carefully in cattle who have received oral electrolytes or are on diets containing anionic salts.

Abdominocentesis

Abdominocentesis can be quite useful in determining the presence of specific conditions, such as uroabdomen and hemoabdomen, and for differentiating between inflammatory and non-inflammatory conditions of the abdomen.

There are four main sites for standing abdominocentesis in adult cattle. Sampling of the left cranial quadrant of the abdomen occurs 5cm caudal to the xiphoid and 5cm to the left of midline; the right cranial quadrant is sampled 20-30cm cranial to the right cranial attachment of the udder and the right and left caudal quadrants sampled 2-3cm cranial to the left and right cranial attachments of the udder. For all methods, the use of a chute and tail jack are recommended.

The site should be clipped and aseptically prepped and an 18ga, 1.5-2" needle introduced perpendicular to the body wall. Alternatively, a teat cannula may be used by desensitizing the skin and body wall with 2% lidocaine, making a skin stab incision and introducing the cannula through the body wall. If a cannula is used, it should be wrapped in gauze to prevent contamination by skin bleeding.

For cytology, the sample should be placed in a tube containing EDTA, while culture samples should be in plain sterile tubes. If the volume obtained will not fill the EDTA tube at least ¼ full, the EDTA tube should be shaken out prior to introducing fluid, as EDTA increases the refractive index of fluids. Samples should then have total protein (TP) determined on a refractometer and air-dried smears made immediately.

Values for normal abdominal fluid in ruminants often overlaps with those associated with pathologic conditions. Therefore, fluid must be interpreted in light of all available information for the case. Abdominal fluid changes composition during the periparturient5 and post-operative6 periods, and the values for calves may be significantly different than cows.7-9

Table 2. Normal peritoneal fluid values for adult cattle.

Volume

1-5mL

Color

Straw

Clarity

Clear

Total Protein

<2.5 g/dL

TNCC

<5000/uL

Neutrophils

<40%

Eosinophils

10-60%

Lymphocytes

<3%

Monocytes

<20%

Fluid of abnormal character or volume may be classified into one of 3 categories.10 Transudates have a TP of <2.5g/dL and a TNCC (total nucleated cell count) of <5000/uL. Transudates may occur in cattle with heart failure conditions, chronic renal failure, parasitism, starvation, maldigestive or malabsorptive disorders or ruptured urinary bladder.

Modified transudates have a TP of 2.5-3g/dL and a TNCC of 5,000-10,000/uL. This fluid may be produced in some normal animals, from portal hypertension from hepatic lipidosis, congestive heart failure, urinary bladder rupture and chronic abomasal displacement.

Exudates possess a TP of >3g/dL and a TNCC >10,000/uL. When degenerate neutrophils compose >80% of the TNCC, septic peritonitis secondary to hardware disease, surgery, vaginal perforation, GI perforation, liver or umbilical abscesses, pyelonephritis or trauma should be considered. Other WBC types may predominate during chronic septic, protozoal, fungal, hypersensitivity or foreign body-induced peritonitis.

Abomasocentesis

Abomasocentesis can be useful for diagnosing Type II Ostertagiasis, abomasal ulcers or to confirm abomasal displacement. This technique may be performed with or without ultrasound guidance. In a study using ultrasound, in 49/50 healthy cows the abomasum was located 10-27cm caudal to the xiphoid and on midline or up to 10cm to the right of it.11 In another study, the site for abomasocentesis was determined to be 12 fingerwidths caudal to the xiphoid and 3-6 fingerwidths to the right of midline.12 The area should be clipped and prepared and an 18 or 16 ga, 2-3.5" needle is inserted through the body wall into the body of the abomasum. Typically, fluid will flow freely, but aspiration may be required.

Fluid obtained should be visually inspected for frank blood, then placed on pH paper or in a pH meter. Abomasal fluid obtained from healthy cows had a mean pH of 2.5 (1.38-4.5) and was negative for occult blood.11 Animals with Type II Ostertagiasis have abomasal pH in the range of 4-7, due to the destruction of gastric acid glands by larvae.

Liver Biopsy

Biopsy of the liver of cattle serves to diagnose both individual and herd-level diseases, including trace mineral deficiencies, and toxic, infectious and metabolic diseases.

Several procedures have been described in the literature, but the author uses a described procedure13 in which a sample is taken at the intersection of a line from the tuber coxae to the elbow on the right side of the animal and the 10th intercostal space. The area should be clipped, prepared and desensitized with 3-5mL of lidocaine. The instrument chosen should be inserted through a stab incision and directed toward the opposite elbow, and the sampling chamber opened and closed. The sample should then be placed in an evacuated tube and chilled for mineral analysis or culture, or placed in formalin for histopathology. Standard rubber top tubes should be avoided for Zn testing; royal blue top tubes have rubber stoppers which do not contain zinc. Samples for mineral analysis may also be frozen.

Instruments used for liver biopsy include manual and automatic Tru-Cut instruments (Bard Medical, Covington VA 30014). When used for liver sampling, it is recommended that 16 or 14ga instruments be selected. While these instruments provide good samples for histopathology and culture, they do not typically provide enough tissue for mineral analysis. If used, at least three samples per animal are recommended to provide a sufficient tissue mass. An alternative is to use the Schackelford-Courtney large animal biopsy instrument (Sontec Instruments, Englewood, CO 80112), which can harvest 150mg of liver tissue per site.13 The major drawback for this instrument is its cost of $400-525.

Liver is the tissue which best represents the trace mineral status of cattle. This is particularly true for Cu, Se, Zn, Fe, P, Co, for which liver is the primary pool or blood levels may be altered by various pathologic and physiologic states. For herd testing, at least 10 animals should be sampled and ideally should be resampled after intervention for any deficiencies. Criteria for the classification of trace mineral status of beef cattle have been summarized.14

Risks associated with liver biopsy include hemorrhage, clostridial hepatitis and peritonitis. In individual animals, the author prefers to perform liver biopsy under ultrasound guidance to avoid puncture of large vessels. This is also useful to ensure capture of focal lesions (granulomas, neoplasias, flukes). If liver abscessation is suspected, fine needle aspiration under ultrasound guidance is preferred to reduce drainage into the peritoneal cavity. This is performed with a 22ga, 6" needle, passed through an 18ga guide needle.15 Suction is placed on the needle with a syringe and the suction released before withdrawing. This aspirate may be placed on a glass slide and stained for microscopy or submitted for culture.

Cholecystocentesis

Cholecystocentesis involves aspiration of gall bladder bile, primarily for liver fluke diagnosis. Two studies16,17 have found sedimentation of bile to be more sensitive (98%16 and 93.4%17) than sedimentation of feces (68%16 and 69%17) in identifying Fasciola hepatica eggs.

The procedure involves the use of ultrasound, as the gallbladder varies in both location and size. Cattle should be well-restrained or sedated. A 3.5 MHz probe is placed at the dorsal aspect of each of the 9th-11th intercostal spaces and used to scan ventrally. Once the gallbladder is identified by its pear shape adjacent to the liver, the site is prepared and a 20ga, 3.5" needle is introduced and 10mL of saline are injected into the gallbladder to stir up sedimented eggs. Then, 10mL of diluted bile is withdrawn.

The sample may be tested similarly to feces for fluke eggs, or simply sedimented in a conical tube overnight in a refrigerator.16 The sediment is then aspirated and examined under the microscope under 10x magnification for the presence of fluke eggs.

Risks associated with cholecystocentesis include making a slit-shaped perforation in the gallbladder due to unsteady penetration of the wall and subsequent bile peritonitis. Studies in live cattle, however, have reported only transient elevated temperatures and the occasional inability to obtain a sample in field situations.16,18

Fecal Occult Blood

Fecal occult blood testing is a useful test to determine fecal blood loss from a variety of GI diseases, including abomasal ulcers, intestinal obstructions, neoplasias and intussusceptions. One report19 studied three fecal occult blood tests in cattle, one of which, guiac solution, was sensitive to a daily blood loss of around 70 mL. Storage the fecal samples for 2 days at room temperature optimized sensitivity. None of the three methods used in this study are available in their stated form. A transit time of 7-19h was determined for abomasal blood.

The author currently uses the Hemoccult card (Beckman Coulter Inc, Fullterton CA 92834). The paper of the card is guiac treated and developed with a hydrogen peroxide and ethanol mixture. These cards are easily run in 3-5 minutes in the field. Cows must have not had a rectal exam prior to this testing.

References

1.  Kleen JL, et al. Dtsch Tierarztl Wochenschr 2004;111:453.

2.  Smith GW, et al. J Vet Intern Med 2004;18:109.

3.  Duffield T. J Dairy Sci 2003;87:59-66.

4.  Oetzel GP. AABP 34th Ann AABP Conv Proc 2001.

5.  Wildon AD, et al. Can Vet J 1985;26:74.

6.  Anderson DE, et al. Am J Vet Res 1994 ;55(12):1633.

7.  Anderson DE, et al. Am J Vet Res 1995;56(8):973.

8.  Burton S, et al. Vet Clin Pathol 1997 ;26(1) :38-44.

9.  Mendes LC, et al. J Vet Intern Med 2005;19(6):899.

10. Kopcha M, et al. Compend Cont Ed 1991;13:519.

11. Braun U, et al. Vet Rec 1997;140(23)599.

12. Wittek T. J Am Vet Med Assoc 2005;227(9):1469.

13. Rogers GM, et al. Bov Pract 2001;35(2):177.

14. Kincaid RL. Proc Am Soc Anim Sci 1999.

15. Hoff B, et al. Bov Pract 1996;30:53.

16. Braun U, et al. Schweiz Arch Tierheilk 1995;137:438.

17. Rapsch C, et al. Int J Parasitol 2006;36:1153.

18. Braun U, et al. Am J Vet Res 1992;53(7):1079.

19. Payton AJ. Am J Vet Res 1980;41:918.

Speaker Information
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Meredyth Jones, DVM, MS, DACVIM
Kansas State University
Manhattan, KS


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