For sick guinea pigs, laboratory diagnostics remain an important part of the diagnostic workup. Due to the non-specific clinical signs, which for owners are only visible at a late stage, guinea pigs are often presented with rather advanced disease. At this point, laboratory diagnostics is usually crucial. It is also commended in cases of poor body condition, inappetence or as part of a preoperative screening in addition to further imaging techniques.

Pre-analytics

As guinea pigs and other small mammals are flight animals and therefore more susceptible to stress than dogs and cats, it is important to have all necessary materials ready before blood sampling, thereby reducing the manipulation time to a minimum.

Due to the small body size and total blood volume (69-75 ml/kg), small blood tubes are recommended. Lithium heparin tubes are particularly suitable for guinea pigs, as it is possible to determine T4 and fT4, besides analysing clinical-chemical parameters and a complete blood count. Lithium heparin blood (1 ml) is therefore a good alternative to EDTA blood and serum if, one expects a small amounts of blood upon sampling.
Furthermore, it is recommended to make blood smears directly in the practice in order for the lab to be able to evaluate the differential blood count in case of transport-related cell degenerations.

Puncture sites

Blood can be collected at different puncture sites using a G20 – G21 needle (with or without cone).
The lateral saphenous vein is most suitable for this. It is located caudally on the lower leg and is clearly visible and palpable (Fig. 1a). A puncture (Fig. 1b) is performed at half height between the femoral canal and the tarsal joint at an angle of 30° to the leg axis.

It is also possible to puncture the vena Cephalica antebrachii, located laterally on the foreleg (Fig. 2). Here, it is recommended to position the leg parallel to the edge of the table.

Haematology

Compared to other species, guinea pigs have fewer, but larger erythrocytes (6.6- 7.9 µm). Haematocrit (0.39-0.55 l/l) is similar to that of dogs; young animal anaemia can also occur. Determination of reticulocytes for the classification of anaemia is also possible in guinea pigs (Kaufhold et al., 2017).

In contrast to dogs and cats, guinea pigs have a lymphocytic blood count. Physiologically, more lymphocytes than neutrophil granulocytes circulate in the peripheral blood. By acute bacterial infections or inflammation, a so-called “pseudo left shift” occurs – a shift from lymphocytic to granulocytic blood count. In this case, a marked leukocytosis as well as the presence of band neutrophils are, compared to other species, rarely seen. By the so-called “stress leukogram” are lymphocytes and granulocytes almost equally distributed.

Neutrophil granulocytes in guinea pigs are also referred to as “pseudo-eosinophils” (Fig. 4) due to their acidophilic staining. These can easily be confused with eosinophil granulocytes, but are smaller in comparison, as their nucleus is less segmented and contains smaller granules.

Eosinophilia occurs, in contrast to rabbits, not only by tissue injury and infections of tissue containing mastocytes (skin, lungs, gastrointestinal tract, uterus), but also by parasitosis.
Guinea pigs can also develop leukaemic lymphomas with severe leukocytosis, which, compared with other species, can easily be diagnosed by a blood examination.

Foa-Kurloff cells (Fig. 6) represent one last haematological feature of this species. Foa-Kurloff cells are mononuclear leukocytes (often lymphocytes, rarely monocytes) with inclusion bodies (Kurloff bodies), which should not be mistaken for intracellular pathogens, due to their appearance. They can make up 3-4% of the leukocytes.

A particularly high number of these inclusion bodies is described during pregnancy and correlates with the oestrogen level. They are most likely equivalent to the killer cells in other mammals and provide a barrier between foetus and mother.

Clinical chemistry

Most laboratories offer so-called Rodent Profiles, which provide a good overview of the individual metabolic status. Often liver, kidney and muscle parameters included, as well as fructosamine, total protein and electrolytes.

Most laboratories offer so-called Rodent Profiles, which provide a good overview of the individual metabolic status. Often liver, kidney and muscle parameters included, as well as fructosamine, total protein and electrolytes.

In order to assess hepatic metabolism, attention should be paid to enzyme changes in GLDH, ALT and AST. In guinea pigs, GLDH is a sensitive enzyme and increases rapidly by acute hepatopathies, although it can be found in kidney cells too. As ALT and AST occur in low concentrations, not only in the liver, but also in other organs, they are not specific for hepatocellular damage. Increased enzyme concentrations, combined with other elevated liver parameters, indicate chronic hepatic disease. Increased AST activity should always be assessed in conjunction with CK, as AST is not only found in the liver, kidney and pancreas, but also in myocardial and skeletal muscle cells.
Although the lipid metabolism in guinea pigs is less active than that of rabbits, they tend to develop hepatic lipidosis. Both AP and γ-GT are slow to react and only increase by chronic conditions. Evaluation of urea and creatinine does not differ from other animal species.

As especially coprophagic herbivores never fast (and should not be fasted before blood sampling), they generally have high glucose levels of 5.0-16.0 mmol/l. However, the physiological fructosamine level is comparatively low at < 271 µmol/l. Glucose is a significant prognostic factor by ileus or stasis. The higher and longer a hyperglycaemia persist, the worse the prognosis for the affected animal. An additional drop in sodium (Na) (< 129 mmol/l) increases the mortality rate by a factor of 2.3 (Bonhevi et al., 2014).

Endocrinology

In contrast to dogs and cats, hormonal diseases in guinea pigs are described in low number. Cases of hyperthyroidism, Morbus Cushing, diabetes mellitus and insulinoma are reported.

Hyperthyroidism

Hyperthyroid animals are often presented at an older age, with progressive weight loss, despite a constant or increased appetite, proliferation of the ventral neck as well as behavioural changes. As the disease progresses, PU/PD, alopecia, inappetence, chronic diarrhoea and cardiac problems can occur.

For diagnosis, T4 should be determined (at least 250 µl of serum or heparin plasma). To exclude differential diagnoses, blood, urine and faecal examinations are recommended as well as a cytological examination of the mass.

Morbus Cushing / Hyperadrenocorticism

Patients with suspected Morbus Cushing are often presented with bilateral alopecia beginning on the flanks and the ventral abdomen, apathy, PU/PD, polyphagia and bilateral exophthalmos. Thinner skin, weight loss and muscular atrophy may also be seen later in the course of the disease.

Diagnostically, a dexamethasone suppression test, as well as an ACTH stimulation test (from blood or saliva) can be used. Due to the pulsatile secretion of ACTH at a high frequency, guinea pigs generally have high baseline cortisol levels in their blood. Any stress, such as blood sampling, additionally increases the cortisol level.
A low-stress alternative is to measure the cortisol level from saliva. The ACTH stimulation test procedure is the same using saliva as from blood. A baseline sample is taken, 20 I.E. ACTH is injected i.m., and the stimulation sample is taken four hours post injection. It is mandatory to use Salivettes for obtaining saliva samples (Fig. 7, Salivette®, Sarstedt).

Transitional cell and prostate carcinoma

In dogs, both transitional cell carcinoma (TCC, Fig. 1) and prostate carcinoma (PCa) are highly malignant neoplasms (GRIFFIN et al. 2018). Both types of tumour are often diagnosed rather late (BRYAN et al. 2007; PANTKE 2018). Consequently, prognosis for affected dogs is bad (CORNELL et al. 2000; HENRY 2003). The median survival time is one year for dogs with TCC and 30 days for dogs with PCa (HENRY 2003; CORNELL et al. 2000). The median age of affected dogs is 11 (TCC) and 10 years (PCa) (CORNELL et al. 2000; KNAPP et al. 2000). For a long time, there was no reliable screening test for the presence of TCC, particularly for breeds with increased genetic risk (certain terrier breeds).

BRAF mutation

The presence of BRAF variant V595E, known from human medicine, was studied for the first time on a large number of tumours in dogs by MOCHIZUKI et al. in 2015. In contrast to people, where the mutation occurs mainly in malignant melanoma, ovarian tumours and colorectal carcinoma, in dogs, MOCHIZUKI et al. (2015) most often found the mutation in transitional cell and prostate carcinomas. It is a somatic mutation in the BRAF gene which only occurs in the tumour cells. It is assumed that this mutation leads to tumour development through permanent activation of the MAP kinase pathway.

BRAF mutation in canine transitional cell carcinoma

In 2018, we succeeded in establishing the method for detecting BRAF mutation in TCC in our laboratory (AUPPERLE-LELLBACH et al. 2018). Specificity was 100% as the BRAF mutation could not be detected in any of the dogs with cystitis, urinary bladder polyp or in those without any clinical and pathological signs. Sensitivity of BRAF mutation detection in TCC was 71%.

Possible sample materials:

• tissue (e.g. biopsies)
• cytological smears (e.g. FNA)
• urine rich in cells (early morning urine)

In the literature, the test has so far only been described for tissue samples and urine. In our studies, we have also succeeded in establishing it for cytological smears of fine needle aspirates and urinary sediments.

Thus, invasive sampling can be avoided by testing spontaneous urine, or there is no repeated invasive sampling necessary in cases of questionable histopathological and cytological diagnoses (poor sample quality, overlapping images of inflammation and neoplasia).

A breed predisposition has been described for canine TCC in Scottish, Fox and West Highland White Terrier (FULKERSON und KNAPP 2015). Another study also showed a predisposition for BRAF mutation in different long- and short-legged terriers (AUPPERLE-LELLBACH et al. 2019). It can thus be concluded that detection of BRAF mutation in these terrier breeds is not only highly specific for the presence of TCC, but also very sensitive (Fig. 2) and can be used as a screening test for the presence of TCC.

In laboratory diagnostics, there are various parameters available for assessing the renal function that provide a good representation of the glomerular filtration rate (GFR).

• Creatinine

• Symmetric dimethyl-arginine (SDMA)

• Urine-specific gravity (USG)

• Protein/creatinine ration (U-P/C)

	pre-renal	renal	postrenal
creatinine	↑↓	↑	↑
SDMA	↑
USG	↑↓	↑↓
U-P/C	↑	↑	↑

The new version of the German Veterinary Pharmacy Act (TÄHAV), which has been in force since 1st March 2018, has raised some questions regarding the treatment of keratoconjunctivitis. Since the TÄHAV came into effect, the main problem for practitioners lies in the fact that, so far, keratoconjunctivitis in dogs and cats has frequently been treated with antibiotic agents that have no approval for use in these animal species in Germany or even contain reserve antibiotics belonging to the  gyrase inhibitor group. We have therefore compared the infectious causes of these diseases with current diagnostic data and show the level of resistance of the pathogens involved to selected agents.

(Kerato)conjunctivitis in cats

Primary infections in cats are mainly caused by feline herpesvirus-1 (FHV-1), closely followed by chlamydia. FHV-1 multiplies in the epithelial cells – including those of the cornea – which is why damage to the cornea (keratoconjunctivitis) with possible subsequent ulceration can occur. These ulcers are almost always secondarily infected with bacteria. Ulcers are a complication that is less frequently seen in chlamydia infections. Chlamydia felis is highly contagious and is shed in eye and nose secretions. Mycoplasma felis is another primary but much rarer cause of conjunctivitis in cats. PCR is the usual detection method for these three primary pathogens. We determine all three pathogens in one eye profile. The material to be submitted is an eye swab rich in cells obtained with a dry swab and sent without transport medium!
Fig. 1 shows the detection frequency of the individual pathogens from the second half of 2017 (total number of samples: 481). PCR detection was positive in 46% of cases, with 39% of all samples tested showing a mono-infection with one of the pathogens and 7% of the samples showing a multiple infection.

Multiple infections most often occurred in combination with mycoplasma, a result consistent with studies in other countries. According to one study, co-infections are associated with more severe clinical signs than single infections. Although FHV-1 is considered to be the most common primary pathogen of the clinical picture mentioned, it cannot be detected in a prolonged infection because the virus is only shed for a maximum of 10 days after infection. Thus, chlamydia have the highest detection frequency in PCR detection, as PCR is often only performed in case of a chronic illness or after the first treatment failed. However, like all herpes viruses, FHV-1 cannot be eliminated, but retreats to different body parts and remains latent there. Triggered by other diseases, stress or corticosteroid treatment, virus shedding can occur again at any time and also lead to recurring clinical signs.

Mild forms of FHV-1-related keratoconjunctivitis are often self-limiting. In severe clinical cases or in case of persistent relapse, however, oral or topical antiviral medication should be used. For chlamydia and mycoplasma, there are no methods established for routine resistance testing. In therapy, antibiotics that have a known effect against these pathogens are used. This also conforms to the TÄHAV. Tetracyclines, chloramphenicol or gyrase inhibitors – used three to four times a day as eye ointment or drops – have a good effect against these pathogens. In case of chlamydia, topical therapy is sometimes not sufficient to eliminate the pathogens in cats, especially if the excretion also occurs via other mucous membranes, e.g. of the genital tract. In these cases, additional oral treatment with doxycycline for 30 days is indicated, especially in multi-cat households. Here, it is recommended to treat all cats of the household with doxycycline. Because of the strong side effects of doxycycline alone, such an approach should be backed up by PCR in any case.

Frequently, the original primary infection is secondarily colonised by facultative pathogenic, aerobic bacteria. Particularly FHV-1-related ulcers are often secondarily infected with bacteria. 866 swab samples of eye swabs from 2017 were bacteriologically evaluated. Fig. 2 shows the pathogen distribution of these samples. Staphylococci take up by far the largest part of detected pathogens, but only 4% of these ocular staphylococci show multiresistance.

In mild clinical cases, empirical topical treatment is often sufficient, since local application achieves a high concentration of active ingredients directly at the site of infection. Cytology can provide information on whether there are indications of inflammation with bacterial involvement. Sampling should be done before the application of cleaning substances or fluorescein. Swabs for bacteriological examination with resistance test must be sent with transport medium! According to a new study conducted by the University of Berlin together with Laboklin, moistening the swab with sterile physiological saline solution improves the detection rate of gram-positive pathogens. The swab should not be placed directly in the pus, as the pathogens contained therein are in a phagocytised form and may therefore not be cultivable.

Although Diff-Quick staining prepared in the practice from a second swab for cytology does not give any indication of the presence of gram-positive or gram-negative pathogens, it does, however, indicate whether bacilli or cocci are present in the preparation. When taking a closer look at the distribution of pathogens, it becomes clear that bacilli always need to be classified as gram-negative and cocci, with the exception of Acinetobacter spp., always as gram-positive. However, Acinetobacter spp. do not play a pathogenic role in conjunctivitis. That is why we have each combined bacilli and cocci in the presentation of the current resistance behaviour, since the cytological results can thus be transferred directly to the empirical selection of an antibiotic (see Figs. 3 and 4).

For cats, there are topical preparations approved in Germany and available on the market for the active substances tetracycline, neomycin and gentamicin (source: Vetidata, December 2018).