There are many different causes of diarrhoea. Intestinal bacterial infections can, for example, induce hypersecretion of fluid into the intestine, which leads to the discharge of watery fluid before or after defaecation in the form of faecal liquid. Malabsorption can also promote diarrhoea. If less electrolytes and fluids are absorbed by the intestinal mucosa due to viral, bacterial or parasitic infections, osmotic imbalance may be caused. This increases the fluid content of the intestinal digesta and softens the faeces. Gastrointestinal symptoms can also manifest themselves as changes in intestinal motility. The equine digestive system bends and becomes narrow in many places. Hard, dry faeces can significantly increase the risk of obstruction. Faulty fermentation causes excessive gas which can lead to the displacement of intestinal sections.

The faecal sample – diagnostic possibilities

Bacteria:

Toxigenic strains of Clostridium perfringens and Clostridium difficile, salmonella, Lawsonia intracellularis and Rhodococcus equi are considered to be primarily pathogenic.

Although clostridia can be grown in anaerobic culture, the clinical signs are usually caused by the toxins. Therefore, toxin detection by means of enzyme-linked immunosorbent assay (EIA) is diagnostically more valuable than time-consuming cultivation, especially since clostridia are also part of the healthy intestinal flora.

Salmonella causes severe, feverish diarrhoea in horses. Foals also suffer from systemic diseases. In adult animals, asymptomatic shedding of the pathogen is possible. Sources of infection are mainly feed and water contaminated by faeces of infected birds, farm animals and rodents. Salmonella can be grown on special culture media or detected by PCR. It should be noted that they are not continuously shed. At Laboklin, testing for salmonella is always part of the bacteriological faecal analysis.

Lawsonia intracellularis, a gram-negative, obligate intracellular bacterium, is the causative agent of equine proliferative enteropathy (EPE). Suckling foals and especially weanlings are affected. Sick animals may suffer from a poor general condition, diarrhoea and colic symptoms, but often “wasting” is the only sign. Detection is done by PCR from faeces.

Rhodococcus equi causes severe pneumonia in foals. Additionally, intestinal disorders can occur. The pathogen can be detected by culture and PCR from tracheobronchial secretion (TBS) or faeces. PCR is more sensitive. Because of interfering factors which may be present in faeces, TBS should be preferred as sample material in this case.

Autovaccines:
Before the production of an autovaccine, a bacteriological examination must be carried out to isolate gram-negative pathogens. An oral vaccine is prepared from the inactivated bacteria and is administered orally to the horse for 20 days. This will help stimulate the production of secretory IgA in the mucous membrane. The use of autovaccines has been particularly successful in treating chronic digestive disorders, especially faecal water.

Viruses:
Rotavirus: Particularly in foals, it plays a role as a diarrhoeal pathogen. Typically, several foals contract the disease within a short period of time. Diagnosis is made by EIA from faeces.

Coronavirus: Mainly adult animals fall ill. Often, fever is the only sign. Morbidity is high but the mortality rate is low. Detection is carried out by PCR from faeces.

Parasites:
Parasitological faecal examinations should not only be carried out in case of digestive disorders, but also at regular intervals in horses that show no clinical signs. To increase the sensitivity of detection, it is recommended to test 3-day pooled faecal samples.

There are 2 different test methods available for the diagnosis of strongyles: Flotation provides a semi-quantitative result. Depending on the number of eggs per field of view, the quantity is indicated as low, moderate and high. In the modified McMaster technique, a defined amount of faeces is floated in a counting chamber so that the parasite stages present can be counted under a microscope. Here, the result is the number of eggs per gram of faeces. This is necessary if the horses are selectively dewormed.
Eggs of large and small strongyles cannot be distinguished microscopically. If they should be differentiated, a larval culture must be established.

Parascaris spp. is the most important endoparasite in foals and yearlings. In adult animals, patent infestation is rare. Detection is performed by microscopy after flotation.

Coproscopic detection of tapeworms only has a low sensitivity. The use of combined sedimentation-flotation techniques can increase the detection rate, but the intermittent shedding of eggs remains problematic.
Serum EIA is superior to faecal pathogen detection due to its higher sensitivity. This can be particularly useful for diagnosis at stock level.

Strongyloides westeri mainly occurs in foals up to 6 months of age; occasionally, adult horses are also affected. The eggs can be detected in fresh faecal samples by means of flotation. If the faecal sample is already several hours old, detection is carried out using the Baermann-Wetzel method.

Protozoa usually only lead to diseases in foals:
Cryptosporidia can be diagnosed by EIA or in a faecal smear stained with carbol fuchsin. Eimeria leuckarti and giardia can be detected microscopically after enrichment – however, for the detection of giardia, EIA and PCR have a higher sensitivity.

Sand:
When hay is fed on sand paddocks or if there is insufficient pasture growth, there is a chance that horses take up too much sand when eating – the risk of sand colic increases. Faecal sand can easily be detected directly at the stable or in the practice. To do this, the horse droppings are dissolved in water, e.g. in an examination glove. Sand settles and can be seen on the bottom. A positive detection of sand is conclusive. However, if no sand is excreted, the presence of sand in the digestive tract cannot be excluded.

Outlook:
Drinking water for animals:
Aside from infectious diarrhoeal pathogens, there are many other causes that can lead to digestive disorders. Monitoring the food and water quality should not be forgotten. Especially if animals do not have access to drinking water and instead drink from open water points or wells, water quality should be tested at regular intervals. At Laboklin, a drinking water profile particularly tailored to the needs of horses will soon be available.

Microbiome:
As in all other mammals, the equine intestinal microbiome also has a close functional connection to the host. A well-functioning digestive system with a well-established intestinal bacterial flora is therefore of essential importance for equine health. Although the causal relationships are still subject to current research, intestinal microbial homeostasis is strongly influenced by factors such as digestive disorders or feed changes. Initial studies on the characterisation of dysbiotic changes of the intestinal microbiome in horses already show promising results. For example, certain members of proteobacteria seem to be significantly overrepresented in horses with faecal water.

In contrast, anaerobic clostridia species and members of the phylum Verrucomicrobia are considerably reduced. In the future, targeted testing of the intestinal microbiome could help to diagnose shifts in the intestinal microbiota and identify predispositions for gastrointestinal disorders. Based on this, it would be possible to prevent or treat gastrointestinal disorders already before the onset of clinical signs by using coordinated treatment concepts (e.g. selected pre- and probiotic agents).

Ann-Kathrin Schieder, Dr. Ronnie Gueta

A quite common type of tumour in mares is granulosa cell tumour (GCT). A diagnosis can often be conclusively made by determining the anti-Müllerian hormone (AMH). AMH is a glycoprotein that influences sexual differentiation during embryonic development and is formed by granulosa cells during folliculogenesis in mature females and by Sertoli cells in testicular tissue in males. Anti-Müllerian hormone can therefore be used as a laboratory diagnostic marker, not only in the diagnosis of tumours but also for various other clinical issues.

For the diagnosis of granulosa cell tumours, AMH is the most sensitive diagnostic marker (98%) compared to inhibin (80 – 90%) and testosterone (50%). Typically, these mares show clinical signs of anoestrus, very low progesterone levels (< 1ng/ml) and a unilaterally enlarged ovary with a striking honeycomb-like appearance on ultrasound. The contralateral ovary, on the other hand, is usually greatly reduced in size. Often, sonographic differentiation from other ovarian diseases, such as ovarian haematoma, teratoma or cystadenoma, is not clearly possible. The determination of AMH can be helpful here, as it is produced in large quantities by the granulosa cells. If the determination of AMH does not provide a reliable result (e.g. if a granulosa cell tumour is beginning to develop), retesting is recommended after 3 – 4 months at the earliest. Furthermore, diagnosis of a GCT by determining AMH is also possible during pregnancy, as the serum AMH level, unlike testosterone or inhibin, remains unaffected in late pregnancy

In the normal cycle of the mare, too, AMH is not subject to any significant fluctuations and, in contrast to testosterone (adrenal cortex), is only produced in the ovary itself. In older mares (> 20 years), a decline in AMH level may occur correlating with a decrease in follicular reserve (primordial follicles).

Determination of AMH can also be useful in stallions. Unlike oestrone sulphate, AMH concentration in male animals is conclusive for the diagnosis of cryptorchidism regardless of age and thus represents a useful biomarker for the presence of testicular tissue. The baseline testosterone level in cryptorchid stallions can be low or borderline and only provides little information when determined solely. Testosterone should therefore always be measured while performing an hCG stimulation test (baseline and stimulation value). Due to its rather long half-life (1.5 – 2 days), AMH should only be determined after a minimum of 2 weeks post castrationem, since serum concentrations will then have decreased to a diagnostically conclusive level. Before these 14 days have passed, testosterone is more suitable as it has a much shorter half-life (approx. 1 h). Very low AMH levels in intact stallions may indicate testicular degeneration or are due to the season (autumn/winter). Age-related testicular degeneration is more common in stallions at an advanced age, but AMH levels do not provide information on the fertility of an animal. Very high levels, on the other hand, in combination with clinical signs, can be an indicator of Sertoli cell tumour and should always be clarified by histopathological examination.

Yet biomarkers do not only play a role in the diagnosis of tumours. If inflammation is suspected, acute-phase proteins such as serum amyloid A (SAA) or fibrinogen may also be helpful to assess the clinically non-visible severity of the inflammation or to estimate therapeutic success.

In general, acute-phase proteins are divided into negative ones, whose concentration in blood decreases during the acute-phase response (e.g. albumin), and positive acute-phase proteins, which, in turn, increase during inflammation. Positive acute-phase proteins are either major (rapid increase within a short period of time by 10- to 1000-fold), moderate (slow increase by 5- to 10-fold with a plateau phase) or minor acute-phase proteins (0.5- to 5-fold increase). The formation of acute-phase proteins is initiated during the acute-phase response of inflammation. This is an early, non-specific systemic reaction to tissue damage from infection (bacterial, viral or parasitic), trauma or neoplasia. In addition to local and systemic effects, the release of inflammatory mediators, such as pro-inflammatory cytokines, stimulates the synthesis of acute-phase proteins in the hepatocytes of the liver.

In horses, determination of SAA as major acute-phase protein is increasingly used in clinical laboratory diagnostics. SAA is an apolipoprotein which, on the one hand, chemotactically recruits inflammatory cells into an inflamed area, but on the other hand suppresses lymphocyte proliferation. In healthy horses, serum SAA can only be detected in very low concentrations. If a noxious agent is present, there is a very rapid (within 6 – 12 h) and strong increase in concentration (100- to 1000-fold) and also a very rapid decrease when the noxious agent is removed (within 12 h). SAA is a very sensitive marker of early inflammatory processes, which can provide important assistance in diagnosis, prognosis and monitoring. Especially when quick action is essential, as for example in foal medicine, SAA levels can be measured to detect septicaemia, bacterial pneumonia or septic arthritis at an early stage. Serum levels of SAA can also help to differentiate between bacterial and viral infections, but should always be interpreted in the context of the general clinical examination and other laboratory parameters, as SAA is a non-specific acute-phase protein. Slightly elevated levels can be measured after stress, transport, vaccination or heavy exertion. Furthermore, increased SAA should never be the only criterion for antibiotic treatment. The white blood count, clinical chemistry and other markers of inflammation and, of course, clinical and bacteriological examinations should also be included.

Fibrinogen, which is a moderate acute-phase protein, is also used as a diagnostic tool in equine medicine. Compared to SAA, it only increases by 1- to 2-fold within the first 24 h after the noxious agent and peaks after approx. 48 h. Fibrinogen may remain elevated for a few weeks and is therefore not as sensitive when monitoring. Fibrinogen can only be determined from citrate plasma.

A quick and early diagnosis is not only relevant in acute inflammation, but also for organs with a low regeneration rate such as the cardiac muscle. A useful biomarker for acute myocardial necrosis is cardiac troponin I, which can be used as a specific cardiac parameter in horses. Cardiac troponins are proteins that play a role in regulating the myocardial contraction and relaxation. If cells are damaged, increased serum levels of cardiac troponin can be detected after approx. 5 – 7 h. Causes may be viral or bacterial, congenital heart diseases, deficiencies, toxins or neoplasia as well.

So overall, several different, very sensitive biomarkers are available in clinical laboratory diagnostics. Separately or in combination, they can provide suitable assistance in the practice in terms of diagnosis, assessment of prognosis and to predict therapeutic success as well as to support decisions on treatment.

Svenja Möller

2.2 Predisposing factors for the occurrence of non-infectious endometritis are anatomical changes of the external genitals, e.g. vulvar cleft, scarring after difficult foaling, a cranially sunken anus as a result of reduced body fat, etc., which facilitate the entry of air or urine into the vagina or even the uterus.

The most common reason for non-infectious endometritis, however, is breeding/insemination which causes an inflammatory reaction of the endometrium (breeding-induced endometritis). Physiologically, it is a transient inflammation which subsides within 48 hours due to effective uterine clearance.

Yet, some mares are more susceptible to developing persistent breeding-induced endometritis (so-called “susceptible mares”), so that after contact of the endometrium with the semen, inflammation occurs with massive accumulation of fluid which cannot be eliminated from the uterus. Thus, persistent endometritis develops, which leads to early embryonic loss.

3 Laboratory diagnostics Bacteriological, cytological and molecular genetic examinations:

If, during the special clinical-gynaecological examination, vaginal discharge or traces of secretion in the area of the external genitals are detected, which indicate an exudative inflammation in the genital tract, it is recommended to take a sterile endometrial swab and to examine cytological samples.

Taking a sterile endometrial swab for bacteriological examination is best done using instruments such as a speculum, cervical forceps and swab collection systems, as this method has the lowest risk of contaminating the swab with bacterial flora of the vagina or the external genitals. Furthermore, this method ensures a high diagnostic reliability of the bacteriological examination. Immediately after collection, the swab should be put into a transport medium (Amies medium) and labelled to avoid any confusion. Samples should be cooled and transported to the laboratory overnight. After collecting the endometrial swab, it is advisable to take a sample of the endometrium for cytological (and possibly histological) examination.

For the detection of Taylorella equigenitalis, two swabs (in Amies medium with charcoal) must be taken from the clitoral fossa and the clitoral sinus (according to Directive 92/65/ EEC), but not from the endometrium.

It is always best to submit the swab immediately after sampling; in general, no more than 24 hours should elapse before starting the bacteriological examination and no more than 48 hours for a PCR test. Please note that when both methods are combined, two separate swabs must be submitted, one for the PCR test and one for the cultural examination.

Cytology provides information about the character of the secretion. The advantage of this method is that it is quick, easy and noninvasive. Using a double-guarded collection system and a cytology brush, secretion and cells are collected from the surface of the endometrium and rolled onto a slide which is air-dried and microscopically evaluated in the laboratory using Diff-Quik staining. If large amounts of mucus are present in the smear, it may be an indication of mucometra. The detection of neutrophil granulocytes and perhaps also of bacteria or fungi indicates purulent inflammation and, thus, an infection. The advantage of combining a bacteriological and a cytological examination is a significantly increased diagnostic sensitivity as well as the possibility to better assess false negative bacteriological results (e.g. due to nonrepresentative sampling).

The examination methods presented so far are suitable for identifying catarrhal-purulent endometritis as the sampling techniques are non-invasive.
By contrast, inflammatory processes which are not associated with exudation of leukocytes into the uterine lumen, can neither be diagnosed clinically nor by bacteriology or cytology.

Endometrial biopsies (histology)

For a comprehensive assessment of chronic and/or deep endometrial inflammatory processes, the histopathological examination of endometrial biopsies is the method of choice. In barren mares, it is therefore recommended to do a biopsy combined with a bacteriological examination at the beginning of the season.

Tissue samples of the endometrium are collected vaginally using biopsy forceps. The endometrial sample is immediately placed in 10% buffered formalin (Fig. 1). Even though this is a (slightly) invasive method, mares usually show no reaction to the sampling procedure, as the endometrium is a tissue with a high regenerative capacity.

During the histopathological examination of the biopsies, the degree, character, site, extent and timing of the inflammatory response are recorded. First of all, this allows for a targeted analysis of the cause (e.g. purulent inflammation – bacteria/fungi; eosinophilic inflammation – semen diluent, flush, foreign bodies; lymphoplasmacellular inflammation (Fig. 2) – chronic local immune response), and secondly, these criteria are relevant for an epicritic evaluation of the lesions in terms of treatment success and, ultimately, the prognosis of fertility.

Fertility prognosis according to Kenney & Doig (1986) and according to Schoon et al. (1992 and 1997):

Inflammatory lesions of the endometrium are included in the categorisation according to Kenney & Doig (1986), but the clinical picture of the inflammatory alteration, its histopathological character as well as its reversibility are not taken into account. However, since these factors are essential for a comprehensive interpretation, they will be discussed in more detail in the epicritic evaluation of the results (Schoon et al. 1992 and 1997) (see Fig. 3).

The categories according to Kenney & Doig (1986) are correlated with the statistical probability that the mare conceives and successfully delivers the foal (see Tab. 1).

Category	Foaling probability
I	80 – 90%
IIa	50 – 80%
IIb	10 – 50%
III	< 10%

Tab.1: Categorisation based on histopathological findings on the endometrium according to Kenney and Doig (1986)

The categorisation represents a current assessment of the expected breeding success at a given time; however, it is important to note that – depending on the type of endometritis and treatment success – the category can be improved (see Fig. 3).

Therefore, the age of the mare, the time of barrenness, the site of the endometritis and its character are also taken into account in the epicritic evaluation of the histopathological findings. The aim is to provide practicing colleagues as much diagnostic information as possible in order to decide whether or not targeted treatment has a chance of success.

Dr. Kathrin Jäger

Pathogenic bacteria in breeding hygiene:

The following bacteria are classified as pathogenic when testing the breeding hygiene; treatment is also recommended for clinically healthy mares before they are bred:

• ß-haemolysing streptococci
• Staphylococcus aureus
• Escherichia coli var. haemolytica
• Klebsiella sp.
• Raoultella ornithinolytica, former: Klebsiella ornithinolytica
• Pseudomonas aeruginosa
• Actinobacillus equuli
• Bordetella bronchiseptica
• Rhodococcus equi

If other pseudomonads or Escherichia coli (without haemolysis) are present, treatment is also recommended before breeding if bacterial count is high and they can be detected in pure culture. The microbiological findings should always be assessed in combination with clinical changes during gynaecological examination.

Overview on breeding hygiene examinations in 2019:

Swabs from female horses were evaluated, which were sent to LABOKLIN in 2019 by veterinarians practicing in Germany for testing the breeding hygiene.
Pathogenic bacteria were detected in approx. 26% of the submitted samples. In 1%, a high level of Escherichia coli was present in pure culture (Fig. 2).

As expected, ß-haemolysing streptococci formed by far the largest group with about 84%, followed by Klebsiella sp. and Escherichia coli var. haemolytica (Fig. 3)

Although Staphylococcus aureus was only the fourth most frequently detected pathogen with 2.6%, it is worth mentioning that 39% of the isolates were resistant to methicillin. Pseudomonas aeruginosa was isolated to a similar extent. Raoultella ornithinolytica and Actinobacillus equuli were very rarely present. Rhodococcus equi could only be grown once. Bordetella bronchiseptica was not isolated.

The mycological examination:

If no pathogens can be detected in the bacteriological examination of a mare with clinical signs or if whitish coatings appear on the mucous membrane, it may be advisable to request an additional mycological examination.

Usually yeasts of the genus Candida are detected. Among the moulds, the genus Aspergillus is the most common.

A predisposing factor for an infection with yeasts is, above all, previous long-term antibiotic treatment.

As sample material, a swab with medium is also suitable here. The test takes up to one week.

Taylorella equigenitalis:

Contagious equine metritis (CEM) is a notifiable venereal disease caused by the bacterium Taylorella equigenitalis. While infected stallions are usually asymptomatic carriers, mares suffer from endometritis and fertility disorders, but inapparent infections also occur.

According to Council Directive 92/65/EEC, swab samples must be taken from the mare from at least two sites – the clitoral fossa and the clitoral sinus. In addition to culturing the organism, PCR is available as another suitable test method. Regardless of the method, only swabs with added charcoal (Amies transport medium) are to be used.

In the laboratory, the culture should have arrived and be started no later than 24 h after sample collection (48 h after sampling for cooled transport). For PCRs, no more than 48 h should elapse between sampling and starting the test.

Culture and PCR are not only available as single services, but also in more economical profiles in combination with the test for breeding hygiene. Please note that a separate swab is required for each method.

The cultural examination takes 7 days (2 weeks for exports to Canada; 3 weeks for exports to Norway). PCR offers the advantage of a faster diagnosis within 1 – 3 working days.