Clinical Pathology

According to McLaughlin and Fish, there are important species differences in the reticulocyte count. The rabbit has 2-4% reticulocytes, which increases with repeated blood samplings and can reach 40% in hemolytic anemia caused by phenylhydrazine{3749}.

The rabbit’s neutrophil stains differently with Romanowsky stains due to the presence of two types of intracellular granules. There are smaller azurophilic granules and larger granules that may stain darker red. For this reason, rabbit PMNs are often called heterophils or pseudoeosinophils. Some rabbits have the Pelger-Huet anomaly, a condition in which granulocyte nuclei are hyposegmented. In genetically affected rabbits this is associated with high mortality, although a few Pelger-Huet cells are an incidental finding in animals without the genetic anomaly.{3749}

Basophils are found routinely in circulating rabbit blood, a unique feature of the species. Although values of 2-5% are routinely measured, this has been reported to be much higher.{3749}

Dogs: The dog erythrocyte is high in sodium and low in potassium, unlike the RBC of rats, man or NHPs. If the serum sample is hemolyzed this will affect the serum chemistry. As in diabetic people, glycosylated hemoglobin can be measured in the dog as an indicator of long-term glucose control.{3754}

Sheep and goats: Goats have small RBCs with low MCV (15-26fL vs. 28-40fL in sheep). Their erythrocytes are also prone to hemolysis, so Vacutainers should not be used for blood collection. Inflammation in sheep and goats often produces differential shifts in the WBC while maintaining normal total counts.

Hemolysis can also be caused by several conditions, including plant toxicosis (Brassica, which is kale or canola), RBC parasitism (Anaplasma, Eperythrozoon ovis, Babesia), IV injection of hypotonic or hypertonic agents, bacterial toxins (Clostridium perfringens type A, C. haemolyticum, Leptospira interrogans), water intoxication, or immune-mediated destruction of opsonized erythrocytes (seen in parasitemia, penicillin administration, or giving bovine colostrum to neonates). Copper toxicity is most often seen in animals that are overfed copper, and suffer some stressful event. Goats are more tolerant of excess copper than sheep. Some sheep breeds (esp. Suffolk) are highly sensitive to copper toxicosis.

Parasitism, opsonization, and plant toxicity usually results in extravascular hemolysis, in which damaged RBCs are removed by the reticuloendothelial system. Anemia, pallor, weakness, depression, icterus and dark urine are the signs. Intravascular hemolysis, resulting usually from bacterial toxins, changes in plasma osmolality and copper toxicosis, cause the additional signs of hemoglobinemia and hemoglobinuria.{4514}

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Serum chemistry

Mice: In the mouse, strain differences exist in selected chemistries. Age may affect calcium more than electrolytes. Strain may affect complement, cholesterol, and serum protein. Gender affects many analytes in the mouse, as does the stage of the estrous cycle. Diet effects are also known in mice; for example, mice do not have carnosine or anserine. Since carnosine is a resource for histidine, the mouse may be more sensitive than other mammals to histamine. At least 48 hours should be allowed after shipment to mitigate any effects upon the immune response in the mouse.{3750}

Pregnant mice produce three proteins (PAMP-1, PAMP-2 and PAMP-4) corresponding to human proteins. These can be detected using a sensitive line immunoelectrophoresis test. Serum sodium is somewhat higher in mice than other mammals. Calcium may be either free or albumin-bound in serum; in mice with hypoproteinemia due to dehydration or glomerulonephritis, calcium may be lower. The measurement method for alkaline phosphatase is important in mice, as when the Hausamen technique is used AP is detected only in kidney and intestine. Using a p-nitrophenyl phosphate hydrolysis technique, however, there are 2 isoenzymes of AP in mouse liver. These liver isoenzymes may change during mouse hepatitis virus infection. Alanine aminotransferase (ALT) is found in many mouse tissues, and is a "leakage enzyme" in that rising values signify cellular damage. It is particularly useful in detecting hepatocellular damage, with 11,000% increases in mice infected with MHV. Aspartate transaminase (AST) is also a leakage enzyme found in many tissues, particularly murine heart and liver. In the liver it is distributed mostly in periportal hepatocytes. Mouse and man both have 5 isozymes of LDH, so destruction of specific tissues is associated with tissue-specific activity in the blood. Creatine phosphokinase (CPK) is increased in mice and other species by skeletal muscle damage, as it is located there in highest concentration; CPK maintains levels of ATP by breaking creatine into creatine phosphate + phosphate.{3750}

Rats: Of all the disciplines using rats as experimental animals, toxicology is among the largest. Such research depends upon both longitudinal data including chemistries, and histologic analysis of tissues postmortem. Hence, much is known about rat serum chemistries. One unique feature mentioned with reference to rats is that alkaline phosphatase (AP) is found mostly in osteoblasts, kidneys and intestine. Male rats have higher serum AP than females.{3751}

Guinea pigs: Guinea pigs, rabbits and NHPs are the only common species that produce GGT, released during clotting. Guinea pig GGT is only 1/6 that of NHPs. Alanine aminotransferase is not a useful indicator of liver disease in the guinea pig, as its total activity in hepatocytes is very low. Cholesterol is synthesized by the intestine in the guinea pig and man, compared to other species which synthesize it in the liver. When fed hypercholesterolemic diets, the rabbit, guinea pig and prairie dog will respond; the rat, dog, NHP and man do not develop high cholesterol in response to the diet. Dietary levels of cholesterol as low as 0.1% may induce biochemical changes in the guinea pig. Another major species difference is in the structure of pancreatic insulin, which differs from other species by 1/3 of its amino acids. To compensate, guinea pigs produce far more insulin and have more tissue receptors. These receptors do not require chromium for binding, and so the guinea pig does not require as much dietary chromium as other species.{3752}

Hamsters: Glucose is higher during periods of hibernation, and in circadian rhythm at the onset of light. Some lines of Chinese hamster have a high incidence of diabetes mellitus, with glucose as high as 500mg/dl. These hamsters are also hypercholesterolemic. Normal cholesterol in hamsters is among the highest reported, from 112-210 mg/dl. Total protein, however, tends to be low (4.5 ± 0.73 g/dl). In a study of hamsters with viral hepatitis, alkaline phosphatase was a better indicator of liver damage than bilirubin or ALT. Thyroid hormones have received much attention because of their involvement in hibernation. Chronic short photoperiod decreases TSH, T₃ and T₄; on the other hand, cold conditions increase T₃ and decrease T₄. Normal hamster cortisol levels are low compared with other species, ranging from 0.38 m g/dl in females and 0.45m g/dl in males.{3753}

Rabbits: In rabbits, some analytes are more affected by stress and handling than others. Glucose and free fatty acids may increase with stress, while insulin and glucagon do not. In general, enzymes that come from muscle (LDH, AST, CK) may increase in a rabbit stressed by handling or invasive collection procedures. Enzymes that come from visceral sources (ALT, cholinesterase, Alk Phos) are less affected.{3749}

Dogs: Physiologic lipemia is present in the dog more than other species, and may make the sample unusable. Lipemia that persists after a 12-hour fast may be due to diabetes mellitus, acute pancreatitis, liver disease, and congenital dyslipoproteinemia. {3754}

NHPs: Uric acid, a poor diagnostic tool in animals, reflects an interesting phylogenetic progression in nonhuman primates. In man, uric acid is the end product of purine metabolism. In subprimate mammals, uricase (urate oxidase) in the liver converts uric acid to allantoin. Prosimians have high levels of uricase and low serum uric acid. New World monkeys have low uricase levels and high uric acid. Old World primates have high uricase levels and low uric acid. {3760}

Glucose tolerance testing must take into account the effects of handling stress; the hormonal response may impair glucose clearance. Ketamine impairs glucose absorption from the intestine. {3760}

In NHPs, ALT is not as specific for hepatocellular injury as in the dog, rat or mouse; however it is sensitive in its indication of injury. Note that leakage enzymes denote hepatocellular injury, not function. AST levels in NHPs increase only when there is necrosis, not so much with cellular injury. Amylase values are 50-150U/l in all NHPs studied except for Rhesus macaques, in which it is 800U/l, comparable to levels in the dog and cat.{3760}

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Serologic Testing

Hemagglutination Inhibition (HAI)

Several viruses have hemagglutinins on their surfaces that bind red blood cells. When present and not inhibited, agglutination occurs; when present and coated with antibody, the agglutination does NOT occur. Viral antigen is incubated with serial serum dilutions in a microtiter plate. If the serum sample is positive for viral antibody, it binds to the hemagglutinins on the test antigens and INHIBITS agglutination; the added red cells fall to the bottom of the plate and form a button. Therefore, the presence of a button indicates a POSITIVE test. In the negative test wells, there is a feathery, diffuse appearance on the slope of the well, caused by agglutination of the red cells. The major advantage of HAI is that specificity is VERY HIGH, however, sensitivity is low and not all viruses are hemagglutinating.{4558}

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Indirect Fluorescent Antibody (IFA)

This assay is used to detect antibodies in serum. Sensitivity is high and specificity is moderate to high. The assay is relatively inexpensive but interpretation is subjective.{4562}

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PCR

PCR is a technique that amplifies a small amount of DNA into a large amount by repeating three steps: denaturation, annealing, and synthesis. First, the DNA is denatured by heating to 95°C. Second, oligonucleotide primers are annealed. Finally, Taq DNA polymerase (derived from Thermus aquaticus, a heat-stable bacterium) adds complementary bases to the single strand template DNA. This cycle is repeated 20-30 times. The results are visualized with gel electrophoresis and ethidium bromide staining.{3924}

Southern blot analysis is an alternative technique, but PCR has several advantages:

1. high sensitivity due to exponential amplification
2. high specificity due to specificity of primers
3. rapid results, in one working day

Disadvantages:

1. small amount of contamination can produce false positive results
2. expensive
3. inhibition of PCR reaction can lead to false negative results

Reverse Transcriptase PCR

This procedure is used to detect viruses with an RNA genome, and RNA transcripts. First, template RNA is isolated from the sample. To generate cDNA, a primer is annealed to the template RNA; this template can be gene-specific, random or oligo-dT primers for mRNA. Reverse transcriptase is added to synthesize cDNA. Next, the template RNA strand is removed with RNAse H, and the resulting single-stranded cDNA can be used for amplification. The standard PCR reaction consisting of addition of an oligonucleotide primer to cDNA, Taq polymerase to add complementary nucleotides to produce ds cDNA, and then the 3-step process of denaturation, primer annealing and extension are repeated. The final product is visualized using ethidium bromide agarose gel after electrophoresis.

Alternative tests include Northern blot and RNAse protection assays. RT-PCR  advantages are similar to PCR, i.e. high specificity, high sensitivity, and speed. In addition to the same disadvantages as PCR, RT-PCR does not detect the final protein product, only the RNA transcripts.{4137}

A problem with using RT-PCR to screen cell lines for contamination with LDHV surfaced in a letter to Comparative Medicine in 2000. Neil Lipman reported getting false-negative results using RT-PCR which was due to inhibitory factors present in pooled sera from mice. The interfering substances could be minimized by dilution. LDHV was undetectable in infected cells that were the origin of the contaminant. Another potential problem was the use of the selected probe; when a new probe was designed the results were better. He urged caution in testing transmissible cell lines and biologics for LDHV contamination. In this case they had confirmed contamination by inoculating 3 SWR mice with one of two doses of cells, and had successfully induced infection by passaging their serum to fresh mice.{4193}

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Fluorogenic 5' Nuclease PCR (Real-Time PCR)

In this assay, an exonuclease enzyme that works from 5' to 3' releases a fluorescent dye that can be detected from a special DNA (or RNA) probe. The result is a graph depicting the amount of fluorescence at the end of each thermal cycle, which is proportional to the amount of PCR product generated. The assay is very sensitive and specific (although false-positives from sample contamination are still a risk), quantitative, and requires no post-PCR processing such as gel electrophoresis.{4548}

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Ribonuclease Protection Assay

Used to quantitate simultaneously several RNA species in a single sample. Useful to study and quantify gene expression. A cDNA sequence to the RNA to be quantitated is replicated with DNA-dependent RNA polymerase. Multiple copies of anti-sense RNA are made, with radiolabeled nucleotides. This RNA is mixed with the sample RNA to make hybridized dsRNA. Then ribonuclease is added, which digests away the ssRNA and leaves the dsRNA alone. The labeled dsRNA is then run on a gel and can be quantitated by measuring the probe radioactivity. The technique is sensitive, specific, requires a lot of sample RNA, is not as sensitive as RT-PCR, and is tedious and time-consuming.{4500}

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DNA Microarray

This complex analysis is used to identify and quantitate the simultaneous expression of thousands of genes. Differential gene expression between control and experimental groups is easily compared. In laboratory animal medicine the technique is used for genetic monitoring, and has potential for use in disease diagnosis.

Thousands of cDNAs can be placed on a single slide. cDNAs from the samples are labeled with different-colored tags. Alternative assays are quantitative competitive RT-PCR, Northern blot, and RNAse protection assays.{4546}

Urinalysis

Mice excrete very small amounts of highly concentrated urine at a time, made possible by long loops of Henle and giant vascular bundles associated with them. The mouse can concentrate urine to 4300 mOsm/l, compared with only 1160 mOsm/l in man. Mouse urine has large amounts of protein, including taurine and creatinine.{3551} This may have relevance for the high incidence of allergy to laboratory animals in people.

In the dog (and perhaps other species), measurement of BUN and creatinine are relatively insensitive methods of assessing renal function, increasing only when 25% of functional nephrons are left. Changes in urine specific gravity and/or osmolality in response to water deprivation are better indicators of renal function. There are three classes of liver dysfunction: (1) leakage enzymes such as ALT and glutamate dehydrogenase, (2) liver function indicators such as bilirubin, ammonia, prothrombin and fibrinogen, and (3) impaired bile flow or function, indicated by GGT. Neither amylase nor lipase are specific for acinar pancreatic cell injury, but lipase has greater diagnostic value for pancreatitis in the dog, in contrast to man. Cholesterol is increased in hepatocellular disease and cholestasis, as well as in hypothyroidism. Pseudohyperparathyroidism is caused usually by cancer, especially lymphoma and adenocarcinoma, possibly due to a tumor-produced peptide with PTH-like activity. Dogs with seriously impaired renal function exhibit renal secondary hyperparathyroidism, in which failure of the renal system to excrete phosphorus stimulates the parathyroids to release calcium and phosphorus from bone.{3754}

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