The keeping of reptiles in captivity is often undertaken with the aim of breeding the species in question. A fundamental prerequisite for successful breeding is knowledge of the sex of the animals.
Additionally, this information is necessary because same-sex individuals of many species can exhibit territorial behaviour, particularly upon reaching sexual maturity, which in the worst cases may result in aggressive encounters.

Although many reptiles, including numerous snake species, display clear sexual dimorphism, this is rarely apparent in juvenile animals, as it generally only develops over time. In many species, sexual dimorphism becomes fully expressed only upon reaching sexual maturity (Fig. 1). A prominent example is the anacondas (genus Eunectes), in which adult females grow many times larger than males. Another example is Wagler’s pit viper (Tropidolaemus wagleri), in which, in addition to sexual dimorphism, there is also sexual dichromatism—that is, a sex-specific difference in colouration and patterning.

While sex determination in adult individuals of many species is relatively straightforward, it becomes challenging in subadult or juvenile animals.
Correctly identifying the sex of hatchlings is particularly important, as these animals are typically transferred to new keepers at this early stage.

There are a number of manual methods for sex determination, each of which often has its own drawbacks.

 

1. Popping

The so-called “popping” technique involves manually everting the hemipenes of male snakes from the hemipenal pockets (Fig. 2). This method has the drawback that it is often applicable only to very young snakes, as the hemipenes of older individuals can no longer be everted. There is also a considerable risk of injury to the spinal region due to tail bending and the application of pressure immediately distal to the cloaca. Furthermore, there is always the risk of misidentification, as males may be incorrectly classified as females if eversion of the hemipenes is unsuccessful.

 

2. Probing

Probing involves inserting a metal probe into the hemipenes or hemiclitoral pockets just distal to the cloaca. The depth of insertion is measured by the number of subcaudal scales the probe passes. The deeper hemipenal pockets of male snakes allow for greater probe insertion. The main drawback is that probing very small snakes is simply not feasible due to the risk of injury, such as perforating the hemiclitoris of females (Fig. 3). There is also always a risk of misidentifi-cation, which can occur in either direction.

 

3. Visual Inspection

Certain characteristics can indicate a snake’s sex without further manipulation. These include, for example, tail shape or visible sexual traits, such as hemipenal pockets on the dorsal scales of colubrids (Fig. 4). However, these methods require considerable experience, are highly unreliable, and should be applied with great caution.

Inappropriate husbandry and diet are among the main reasons for the presentation of birds and reptiles in veterinary practice. One of the reasons for this is that it is often difficult to imitate or replace all conditions of the natural habitat from which a species originates. Many of the avian and reptile species kept as pets originate from tropical regions with a completely different climate and significantly more sunlight, which is crucial for the production of vitamin D. The diet is also generally different; in the wild, animals have access to a much broader variety of food items and many plants or animals are either unavailable or unsuitable in captivity (Fig. 1). The nutrient content of the diet is therefore often significantly different in managed care. Seeds, for example, are typically low in vitamins A and D, but also in calcium (Harper and Skinner 1998; Koutsos 2016). There is also a similar problem with insects, which should be gut-loaded with high-quality feed before being offered to animals (Boyer and Scott 2019). Carnivorous species should preferably be given whole prey, because the nutrient content in the organs such as the liver is naturally different from that available in pure muscle meat.

 

Diagnosing kidney disease in birds, reptiles, amphibians, and fish can be challenging, and there are fewer tools available for laboratory evaluation compared to those for mammals. The blood analytes associated with the kidney also differ depending on the habitat, diet, and in some cases on additional factors including season. There are therefore no standard kidney analytes that can be used in all species, and reference intervals must also be interpreted carefully. In this newsletter, we will discuss the individual relevant analytes and factors influencing blood values to provide an overview of how laboratory testing can aid with your kidney patients.

Let’s start with the differences in the ecology of various species and why this has such an influence on the kidney-associated analytes. For animals adapted to arid habitats, excretory products must be removed from the system using as little water as possible. In lizards, snakes, tortoises, and birds from such habitats, this is uric acid. Some amphibians, such as some tree frogs, also produce uric acid.
In geckos and agamas, for example, this is found as dry white crystals in their droppings (Figure 1).
The more humid the natural habitat of the animal, the more the end product of purine metabolism changes from uric acid to urea, which is excreted with more water causing excretions to change from solid to pasty to liquid. This is the case e.g. in pond turtles, alligators, and some amphibians.
Animals that live completely in water also excrete ammonium, e.g. sea turtles, some crocodiles, fish, and amphibians. The end product of purine metabolism therefore differs depending on the ecology and physiology, and the most suitable analytes to evaluate kidney function vary accordingly.

The diagnosis of thyroid hormone disorders plays a significant role in small animal medicine, especiall in dogs, cats, and guinea pigs. However, birds and reptiles can also exhibit thyroid disorders, often due to causes like thyroiditis or iodine deficiency.
Hypothyroidism has been reported in various bird species, including grey parrots (Psittacus erithacus), amazons, macaws, parakeets, and others.
For instance, a case involving a hyacinth macaw (Anodorhynchus hyacinthinus) revealed ulcerative dermatitis and valvular endocarditis (Huynh et al., 2014). Additionally, feather loss, obesity, hypercholesterolemia, and non-regenerative anemia have been documented in affected birds (Oglesbee, 1992). On the other hand, hyperthyroidism, often resulting from hormone-producing tumors, is rare in birds.

Tortoises with hypothyroidism exhibit anorexia, lethargy, and myxoedema of the skin around the head, neck, and forelimbs. Due to the thyroid gland’s location at the base of the heart, swelling in the neck area is rarely observed. Giant tortoises, such as the Galapagos tortoise (Chelonoides nigra) and Aldabra tortoise (Aldabrachelys gigantea), are particularly affected. The underlying cause is usually an iodine deficiency in the diet or the consumption of goitrogenic plants, such as pak choi, broccoli, cabbage, and soya. A case of hyperthyroidism has also been documented in a green iguana (Iguana iguana), triggered by a thyroid adenoma. This condition led to weight loss, polyphagia, hyperactivity, increased aggression, loss of dorsal spines, tachycardia, and swelling in the neck area (Hernandez-Divers et al., 2001).

The diagnosis of hypothyroidism in birds and many reptile species can be particularly challenging, as thyroxine levels (tT4) in the blood are typically very low within the normal range. As a result, they often fall below the detection limits of commonly used analyzers (e.g. < 0.12 µg/dL). In birds, TSH stimulation tests using bovine and human TSH have also shown success. However, routine TSH measurement is not currently possible due to the species-specific molecular structure, and free T4, T3, and fT3 are rarely determined diagnostically.

Laboklin has developed an LC-MS method that reliably measures low tT4 levels in birds and reptiles, with a detection limit of < 0.02 µg/dL. This method has been tested and validated using blood samples from various species of turtles, lizards, snakes, and psittacids and is now available for routine diagnostics under service number 1089. However, due to the large species variability, no reference values can be provided.

PCR check-up when purchasing new parrots

Parrots, which include the popular budgerigars and cockatiels, are commonly kept pets. According to a market research analysis by the German Industrial Association of Pet Care Producers, there were 3.1 million birds in 3% of the German households in 2021. In line with the recommendations of the expert group on the minimum requirements for keeping parrots, these birds are usually not kept as individuals, but in pairs or small groups.

When acquiring a new or an additional parrot, there is a risk that the bird may suffer from a chronic disease and that infectious agents may be introduced into the flock. Laboklin offers PCR profiles for parrots and parakeets, which cover the most common infectious agents and, optionally, include genetic sex determination.
Parrot bornaviruses are the causative agent of PDD (proventricular dilatation disease) in parrots. The disease was first described in 2008. In pet parrots, mainly parrot bornavirus 2 (PaBV-2) und PaBV-4 are detected, with PaBV-4 being the most common. Mixed infections are also possible. Both PaBV-2 and PaBV-4 cause the characteristic chronic neurological and intestinal signs. For other related viruses, the clinical presentation has in some cases not been described yet or is still incomplete.

Typical signs of PDD are the eponymous proventricular dilatation, delayed passage of intestinal contents, excretion of undigested grains and diarrhoea. This may cause digestive disorders and emaciation up to cachexia. In addition, there may be neurological deficits such as ataxias and coordination disorders, epileptic seizures, tremors and lameness. It is also suspected that parrot bornaviruses cause retinitis and blindness, as well as behavioural disorders such as feather plucking and self-mutilation, but this connection has not yet been confirmed in studies. The course of the disease is highly variable and can range from peracute to chronic. Usually, the progress is slow and chronic, recovery is very rare, but not impossible. Asymptomatic carriers of the virus, which shed the virus for life without becoming ill themselves, have been described. The incubation period varies, depending on the virus strain and the parrot species, and has ranged from 3 weeks (in a study with cockatiels) to 9 months in studies. Transmission probably occurs subcutaneously (e.g. claw and bite wounds and other skin lesions); animal experiments have not yet succeeded in transmitting the viruses by oral route. The most reliable way to diagnose PaBV infection is the combination of antibody and pathogen detection. At Laboklin, we offer a comprehensive PCR test for parrot bornaviruses that can detect PaBV-2, PaBV-3, PaBV-4 and PaBV-7. In some birds, only the direct detection of viral RNA is successful, in others, only antibodies are found, while others again are positive in both tests.

However, in many birds, serology is positive, whereas the PCR test is negative, which can be explained by the fact that these viruses are only shed intermittently and, over certain periods of time, cannot be detected directly. Suitable sample materials include cloacal swabs (for PCR, always dry swabs without medium), faeces, feathers, tissue (intestinal tract and neural tissue); during viraemia/acute phases of the disease, EDTA whole blood can be submitted as well.

Turtles and tortoises are popular pets which can become very old when kept properly. Apart from the commonly kept European species, such as Hermann’s tortoise (Testudo hermanni) (Fig. 1), experienced owners also enjoy keeping exotic species like radiated tortoises (Astrochelys radiata) or leopard tortoises (Stigmochelys pardalis). Clinical signs often appear very late and are only very non-specific in chelonians. Thus, in addition to the standard clinical examination and imaging, laboratory testing is the most important diagnostic tool for providing an accurate diagnosis quickly and at an early stage. This is particularly important in late summer for animals from temperate zones, as a detailed diagnosis is recommended before brumation so that a “rude awakening” or no awakening at all during and after brumation can possibly be avoided. The most helpful laboratory tests include haematology and biochemistry, parasitology, molecular biology and serology.

Blood testing in chelonians

Lithium heparin blood is best suited for blood testing in chelonians (Fig. 2). Clinical chemistry is particularly important for monitoring organ function, as hepatic or renal impairments can be dangerous for the animals, especially during brumation.
Depending on the species, gender, season and temperature, there may be changes in the individual clinical chemistry parameters that need to be taken into account. Contamination with lymph causes reduced protein levels, liverassociated enzymes, uric acid levels and a shift in electrolytes. Trauma during blood collection, for example after several unsuccessful venipuncture attempts, can lead to an increase in alkaline phosphatase and creatine kinase. Considering the wide range of normal values in some species, it is advisable that blood testing is also carried out on healthy animals, so that it is easier to compare and interpret the results if the animal is ill.

Kidney diseases

Uric acid, the major end product of protein and purine metabolism in terrestrial species, is the main indicator of kidney disease. There are strong increases in various kidney diseases, gout, renal dysfunction due to bacteraemia and septicaemia as well as in kidney necrosis due to nephrotoxic drugs such as aminoglycosides and sulphonamides. Feeding animal-based food, as it is physiological in some aquatic turtle species, can also lead to an increase in uric acid. Lowered levels are associated with hepatic diseases. Urea only plays a minor role in terrestrial species, as the levels remain within the normal range for a long time even if uric acid levels are high. In aquatic species, urea also is an excretory product of protein metabolism, which is why it plays a greater role in the diagnosis of kidney diseases. In kidney diseases, blood phosphate levels increase as well, but hyperphosphataemia can also be caused by excessive dietary intake, hypervitaminosis D and haemolysis. In young animals, phosphate levels as well as calcium and alkaline phosphatase are physiologically increased as a result of bone growth.

Hepatic diseases

GLDH (glutamate dehydrogenase) is the main indicator of liver cell damage. It naturally occurs in the mitochondria of liver cells and is only released into the blood when the cells are destroyed. Other enzymes also found in liver cells are ALT (alanine amino transferase), AP (alkaline phosphatase) and AST (aspartate amino transferase). However, these enzymes are not only found in the liver, but also in other organs of the body, so they are not very specific and have to be interpreted in combination with other blood test results and physiological conditions. Liver function parameters are substances that are naturally synthesised by the liver and are decreased or increased if the liver function is disturbed. In this context, especially bile acid should be mentioned, though it can also be affected by feeding, bile duct obstruction and dehydration. Other functional parameters are the protein fractions in the blood, which are, however, also influenced by food intake and renal function. For diagnosing steatosis, it may be useful to determine the triglyceride and cholesterol levels in the blood, as these are usually elevated in this context. However, it should be noted that they are physiologically elevated in females during yolk formation (vitellogenesis).

Haematology

By determining the level of haematocrit, anaemia or dehydration can be diagnosed. A haematological analysis can also provide important information on inflammation. Shifts in cell count are less pronounced in reptiles than in mammals and are therefore more difficult to interpret. However, bacterial and parasitic infections as well as stress can lead to an increase in heterophilic granulocytes, too. Eosinophilic granulocytes are increased in parasitic infections and when the immune system is stimulated. An increase in lymphocyte count occurs during wound healing, inflammation as well as parasitic and viral infections. Changes in the morphology of individual cells can also indicate infections. When looking at the smear, parasites or inclusions due to viral or bacterial infections can be diagnosed as well, but they should not be confused with artefacts caused, for example, during the preparation or drying of the smear. Species, gender, age and physiological condition of the animal also influence the total count and the ratio of the different leucocytes. It is, however, also important to note that physiologically there can be strong seasonal fluctuations in cell distribution.

In recent years, our understanding of infectious diseases in reptiles has grown immensely. It is also clear that the connection between infection and disease is often dependent on multiple factors, including pathogen specific factors (e.g. strain specific properties, virulence factors) and host specific factors (e.g. species, age, sex), as well as environmental conditions (e.g. temperature, hygiene, time of year) and coinfections with other agents. Infections in reptiles also often persist, making quarantine particularly important in this group of animals. The diagnosis of infectious agents in reptiles has also made great strides in recent years. The following pages contain an overview of select infectious agents found in tortoises and turtles, snakes, and lizards. The overview is mainly focused on microorganisms (viruses, bacteria, and fungi), although some parasites are also included. If you are unsure what tests or what types of samples are best in a specific case, we are happy to consult with you on cases.

Pet reptiles are frequently presented to the practice for various types of skin lesions. There are many possible causes for these lesions and they are often multifactorial. These include, first and foremost, husbandry issues, such as inappropriate temperature or humidity, unsuitable substrate or furnishings or poor hygiene. A detailed clinical history and an exact diagnosis are essential for successful treatment.

The purpose of this Laboklin aktuell is to give you a brief overview of the most important infectious causes of skin lesions (excl. parasites). Mixed infections are common and many factors influence how infective dermatitis progresses. A difinitive diagnosis often requires tests such as cytology and histopathology combined with direct pathogen detection and interpretation of results in the context of the clinical presentation.

Viral causes

There are a few viruses which are frequently found in association with skin lesions in reptiles. In some cases, they are considered the primary cause, while in others, their role in the development and progression of the disease is less clear.

Iridoviridae

Iridoviruses of the genera Ranavirus and Iridovirus have been found in reptiles. Both have been described in association with of skin lesions, especially in lizards.

Over the last few decades the popularity of reptiles as pets has increased. This trend is clearly reflected in the increasing number of exotic trade shows and the availability of reptiles in many pet shops. The animals are offered at low prices or even as an „all-inclusive set“ and purchased by unknowing pet lovers. Frequently the rude awakenings follow immediately – the reptiles become ill and are presented at the veterinary practice more and more often.

This of course provides a welcome variety in everyday practice life, but it also poses a new challenge to the practitioner.

There are some difficulties arising from differences between reptiles and the common mammals: the physiology of the ectothermic animals, their reaction to medications, available laboratory tests and last but not least a different morphology of blood cells.

Blood sampling

The blood sampling technique varies depending on the patient species (Tab. 1).Techniques such as clipping of toenails or tail tips are rendered obsolete and are no alternative to professional venipuncture.

General guidelines:

– A blood volume of 0.5% up to 0.8% of the body weight can be sampled safely from a healthy, normally hydrated animal

– The anticoagulant of choice is Li-Heparin, as EDTA may cause haemolysis, especially in tortoises and turtles

– Immediate preparation of a blood smear in order to avoid pre-analytical errors like the deterioration or clumping of blood cells

(Tab. 1)

Sample analysisk

There is a fundamental difference between mammalian and reptilian blood cells: In reptiles all blood cells are nucleated, whereas in mammals only leukocytes and erythroid precursors show a nucleus (Fig.2).

In general, haematology analysers first count In general, haematology analysers first count erythrocytes and thrombocytes, then lyse these cells and subsequently count the nucleated leukocytes. Unfortunately the nuclei of reptilian erythrocytes and thrombocytes do not lyse and therefore a reliable and exact separation of the different cell lines is not possible. Hence automated cell counters are not applicable for reptile blood samples.

Blood cell counts are performed by manual methods:

One possibility is the use of a haemocytometer with different staining and solution systems (e.g. the Unopette system or Natt and Herrick’s solution). These techniques are time consuming and results actually show a higher variability than results of automated methods.

Another manual technique is the estimation of leukocytes and thrombocytes using a stained blood smear. With this method the variability of results is also high, but the procedure is rapid and simply performed with a little bit of experience. Using the 40x objective (400x magnification, if required 100x objective/ 1000x magnification) leukocytes are counted in at least 10 fields of the blood smear. The average cell count per field is calculated and multiplied by the square of the magnification of the used objective (i.e. with 400x magnification by factor 1600, with 1000x magnification by factor 10000). The result is the number of cells per μl blood.

A small calculation example:

7 Leukocytes/400x field
7×1600=11200
The leukocyte count is 11200/μl

Thrombocytes are counted in the same way with the 100x objective (1000x magnification) but multiplied by factor 15000.
Furthermore the differential leukocyte count and morphology of the blood cells are obtained by microscopic examination of blood smears.
It is not possible to estimate the erythrocyte number. The packed cell volume (PCV) is determined by microhaematocrit centrifugation as in mammals. Haemoglobin concentration can be measured by haematology analysers.

Blood Cell Morphology

Erythrocytes of reptiles are ellipsoidal and have a centrally positioned nucleus. Depending on the stain used, the colour of the cytoplasm ranges from orange to pink or pale violet. Round basophilic or irregular clear inclusions (Fig. 3) are regularly found in the otherwise homogenous cytoplasm. In general these findings represent artefacts of slide preparation, staining or a prolonged drying period. Slight anisocytosis and polychromasia are normal in most reptiles. Increased numbers of polychromatic (juvenile) erythrocytes are found in regenerative anaemia, during ecdysis and in young animals.

In some cases of regenerative anaemia, massive inflammation or post hibernation erythroid precursors may be found (Fig.4). These are not necessarily a sign of bone marrow disease as in mammals.

Thrombocytes are frequently aggregated on reptile blood smears which complicate the estimation of their number. However, if some aggregates (Fig. 5) are present the thrombocyte number is probably normal. The morphology of reptilian thrombocytes is variable, but they are always nucleated and mostly ellipsoid (Fig. 5). The cytoplasm is usually colourless, but can stain pale blue or pale violet. If the thrombocytes round up (e.g. because of activation) they can easily be confused with small lymphocytes (Fig. 6).

Reptilian leukocytes are classified as heterophils, eosinophils, basophils, lymphocytes, monocytes and azurophils. Classification is occasionally difficult, because the morphology of the blood cells sometimes varies significantly between reptilian species, as well as between subspecies of one species. The differential leukocyte count naturally exhibits a high variation, on the one hand between individuals due to season, temperature and partly gender, on the other hand due to species differences. Some reptile species show a lymphocyte pre-dominant haemogram, i.e. lymphocytes are the most prevalent cell (e.g. bearded dragon, iguana, tortoises), whereas in others granulocytes predominate.

Lymphocytes resemble those of mammals and are easily differentiated from other leukocytes. They have a round nucleus with dark chromatin and only a scant amount of basophilic cytoplasm.

Lymphocytosis occurs during inflammations and viral infections.
The function of heterophils is analogous to the function of neutrophils in mammals. Heterophilic granules stain orange to pink with quick stains. Sometimes granules dissolve and make the cytoplasm appear eosinophilic (Fig. 7, lower left). The differentiation of heterophilic and eosinophilic granules only by their colour is sometimes impossible. However, heterophils are mostly of larger size and the granules are long, while eosinophilic granules are almost always round.

The nucleus of heterophils is round in some species (e.g. tortoises, crocodiles, snakes); other species (e.g. iguanas, chameleons, dragons) show a segmented nucleus in mature cells.

Heterophilia is primarily associated with inflammation (often together with monocytosis). Other causes are stress, paraneoplastic syndrome or leukaemia (uncommon). In severe, acute inflammation animals that normally have segmented heterophils can show a left shift (bands). In extreme cases toxic changes are also visible: loss of physiologic granules, basophilic and/or vacuolated cytoplasm and toxic granulation (Fig. 8).

Eosinophils display bright red or orange, round granules in most reptiles. Some species (e.g. green iguana, bearded dragon) show also small and round, but blue to grey granules. The number of eosinophils varies highly between species (chelonians often show high numbers) and with various factors like season and parasitic infestation (Fig. 9).

Basophils have many dark purple staining granules that sometimes obscure the eccentrically placed nucleus. Using quick stains basophils often show only few granules or appear vacuolated (Fig. 10).

Monocytes have variably shaped nuclei, as in mammals. They have moderately blue-grey cytoplasm that can be clearly vacuolated in activated cells. Monocytosis is present in various inflammatory conditions, especially in granulomatous forms (Fig. 11).

Melanomacrophages are a special kind of monocytes in lower vertebrates that have phagocytized melanin granules. They can be found in inflammatory disease, especially in dermatitis (Fig. 12).

The origin of azurophils is an intensely debated topic. Morphologically the cells look like monocytes with dust-like eosinophilic granules in the cytoplasm (Fig. 13). Some authors classify them as subgroup of monocytes. Other investigators regard them as a separate cell type. In addition, the clinical significance of azurophils is still unclear.