In general, the diagnostic approach to a neonate will not differ from that in an adult except that the small size of the puppy
may preclude some tests, and changes with development may confound the interpretation of others. While I focus on the nervous
system in this discussion, it is important to remember that congenital defects can affect multiple organ systems. A thorough
evaluation is warranted to detect concurrent deficits affecting other systems such as cardiac, urogenital or musculoskeletal.
When assessing an ill neonate, evaluate a hematocrit and blood glucose concentration and, if sufficient samples are available,
perform a complete blood count and serum chemistry profile. Examine a blood smear for evidence of vacuolations or inclusions
since some lysosomal storage diseases will affect leukocytes. Consider blood gas analysis if there is a high anion gap or
low bicarbonate concentration on the serum chemistry profile. Electrolyte abnormalities are common in other neonates and should
be investigated in puppies as well, and thyroid function can be measured to rule out congenital hypothyroidism. Blood ammonia
values are valuable when searching for evidence of portosystemic shunts and will detect other congenital diseases such as
urea cycle enzyme deficiencies. The test must be run immediately after the sample is taken, however, which limits its usefulness
in most practices. Many inborn errors of metabolism produce abnormal byproducts that are excreted in the urine where they
can be readily detected through special assays. Ketonuria in a neonate that is not anorectic could indicate an inborn error
of metabolism. In contrast to blood, where care must be taken with the volume collected from a neonate, urine can be collected
with impunity. Also, thought should be given to collecting whole blood for DNA extraction if a genetic disease is suspected.
There are other diagnostic modalities to consider using in these cases. Imaging of the brain can reveal congenital malformations
such as polymicrogyria, Dandy-Walker malformation or hydrocephalus. Ultrasonography may be able to detect the cerebellar atrophy
of Dandy-Walker malformation through the foramen magnum and dilated ventricles of hydrocephalus through the fontanels if open.
In these patients, magnetic resonance imaging is the most sensitive modality, and because of the small size of the neonate,
a high-field magnet will provide the best images. Keep in mind that myelination will be incomplete so the gray-white matter
distinction will not be as great as in an adult. If electrodiagnostics are performed to diagnose neuromuscular disease or
seizure disorders, the changes with development need to be taken into account.
In many of these cases, postmortem examination offers the best opportunity for a definitive diagnosis, but it is important
to collect data such as serum chemistry profile findings or electrodiagnostics antemortem. Ideally, a pathologist should perform
the postmortem, but this is sometimes impractical. In addition to routine examination of the liver, kidney, lung, etc., depending
on the nature of the condition, brain, spinal cord, peripheral nerve or muscle samples should be obtained for evaluation.
If myopathy is suspected, submit fresh muscle overnight to a laboratory that can process it for immunohistochemistry. Liver
and kidney are very metabolically active tissues that are often used for biochemical studies. Some studies must be done on
brain tissues, but cutting the brain before it is fixed can create artifact. Accessing the brain for laboratory analysis is
easier in these patients. The skull of a neonate is often thin and easily removed with rongeurs or even a side-cutting wire
cutter. The neonatal brain is very soft, however, so care must be taken not to damage it during removal. If more than one
puppy in a litter is affected, one brain could be fixed in toto, while another could be split sagittally and half frozen for
potential biochemical analysis. Consideration should also be given to collecting frozen tissues for biochemical assays or
Gene discovery strategies
Many genetic mutations can manifest as neonatal disease. Development of the nervous system is a complex process, and many
genes are only expressed for a brief period during development. There is a bias for mutations to interfere with that process.
Inbreeding and founder effects can make genetic diseases more common in purebred animals, but genetic disease can arise even
in randomly breeding populations. Littermate and extended family history can help determine whether genetics is playing a
role in the disease. Breed tables can be consulted to see what familial diseases have been reported in the breed; the signs
can then be compared to the case at hand. Individual breed club Web sites can provide a link to information about genetic
diseases and DNA tests available. As with all information on the Internet, a healthy degree of skepticism is needed when evaluating
Modern molecular genetics has provided us with the canine genome map and the tools to identify the genes responsible for hereditary
diseases. Breeders are aware of the value of DNA testing, but veterinarians have the background to interpret the results of
such tests as well as take the lead in developing new tests. Identifying the specific mutation responsible for a genetic disease
can improve therapy and provide breeders the tools to decrease the incidence of the problem. When facing an unknown disease,
gene discovery strategies can be used to identify the gene responsible. Current approaches to gene discovery include the candidate
gene approach, linkage mapping and single nucleotide polymorphism (SNP) association.
The candidate gene approach involves identifying the comparable disease in humans, rodents or other species for which genes have been identified. The
more accurate and molecular the diagnosis in each case, the more refined the list of candidates can be. For example, when
two dachshund littermates with a syndrome of blindness, progressive ataxia and terminal myoclonic seizures were necropsied,
fluorescent microscopy of brain tissue showed fluorescent material characteristic of neuronal ceroid lipofuscinosis (NCL)
within lysosomes. Mutations in eight genes have been identified in humans, mice and sheep with NCL, so these were candidates
for causes of the disease in dachshunds. Electron microscopy showed curvilinear bodies in the dog similar to those found in
humans with one form of NCL. The gene associated with this form of NCL codes for the lysosomal enzyme TPP1, and the affected
dog showed no activity for that enzyme in biochemical assays using frozen brain tissue. Thus, based on electron microscopy
and biochemical data, there was one highly likely candidate. Sequencing the comparable canine gene demonstrated a missense
mutation that led to a premature stop codon truncating the protein prior to the active site. The candidate gene approach can
identify the mutation with a single affected case as was done here, allowing for quick development of a DNA test to identify
carriers as well as a definitive diagnosis in affected dogs.