(This article was presented in Avedila, Expoaviga 2000)

Reproductive problems in small ruminants probably constitute the most important disorder in view of the economic implications in terms of both milk yield and meat production. The present study presents the evidence found in the literature and laboratory concerning the great etiologic variability of reproductive problems in small ruminants. Some data are also presented regarding prevalence, diagnostic techniques, sampling, differential diagnosis, and so on.

Etiology

The etiology of abortive processes in small ruminants is highly varied, and is by no means limited to Chlamydia psittaci, Salmonella spp. and Brucella spp. Table 1 shows a list of the main causal infectious agents described in the literature. It should be taken into account that abortive processes attributable to non-infectious causes (intoxications, hereditary factors, metabolic and nutritional problems, physical factors, etc.) will not be dealt with here.

Table 1. Principal infectious etiologic agents described.

Of all the pathogens cited, L. interrogans and border disease virus are of particular importance due to their high prevalence. They will consequently be dealt with more in detail.

Leptospira interrogans

The genus Leptospira currently includes 10 species, of which 6 correspond to pathogens (L. borgpetersenii, L. inadai, L. interrogans, L. noguchii, L. santarosai and L. weilli), and 4 to saprophytes (L. biflexa, L. meyeri, parva and L. walbachii).

The fundamental taxon is the serovar, which is determined by crossed agglutination testing. In this context, L. interrogans remains the species with the serovars of greatest diagnostic interest.

Serovars can be identified by their characteristic antigenic patterns, identified by means of a monoclonal antibody battery that can provide a rapid and easy identification, particularly if the serovars found in a given area are well known. In this sense, 76% of all strains of Leptospira isolated internationally correspond to 14 serovars, based on the findings of monoclonal antibody tests and DNA analyses (www.kit.nl).

In Spain, leptospirosis seroprevalence as reported in the literature to date varies from one community to another, in the range of 10.4-46.8% (Table 2). Nevertheless, only four serovars have been reported by these sources.

Table 2. Seroprevalence of leptospirosis (10)

All these considerations of Leptospira are important for centering the diagnosis. When a diagnosis of L. interrogans is requested, it is important to know which serovars can be identified. Furthermore, if vaccines are used against Leptospira, it is necessary to know which serovars are included in the formulation.

Border disease

Border disease (BD) is found throughout the world, with prevalences in sheep that range from 5-50%, depending on the country (32); the figure in Spain is close to 18% (27), though herds have been described with reproductive problems in 79-96% of cases (23). These prevalence rates, bovine-ovine interspecies transmission (3,23), the existence of immune tolerant animals that excrete the virus (32), and the lack of effective vaccines have classified BD as one of the most important diseases in small ruminants, requiring special attention and inclusion in the corresponding differential diagnosis.

It is important to point out that in the same way as bovine virus in BVD, BD can involve the existence of asymptomatic carrier animals. During gestation the fetus becomes infected with the virus; these animals survive but their immune system does not recognize the virus as foreign, and no antibodies are therefore produced against it. In this way we have virus-excreting animals that do not undergo seroconversion. The technique used to identify such carriers is detailed below.

Differential diagnosis

Table 3 provides a very simplified account of the distinctive characteristics of the etiologic agents searched for.

Table 3. differential diagnosis.

Although clinicians are able to diagnose the abortions based on their professional experience, it is generally very difficult to clinically distinguish the underlying etiology. Consequently, it is normally necessary to resort to a laboratory for establishing the differential diagnosis in abortive processes.

Diagnostic techniques

The direct techniques detect the presence of the pathogen directly — either by isolating it in culture or by detecting the antigens in host tissues. In general, all pathogens are susceptible to isolation, though Table 4 only indicates the possibility of culture in those cases where it is applied as a routine diagnostic procedure.

However, it is normally not possible and/or not of interest to perform microbiological isolations, in part because of the technical difficulties and costs involved, and in part due to the time investment required. The direct techniques locate the antigen (or DNA) in the tissue of the submitted sample. The antigen is revealed by directing antibodies against it, and the specificity of the result obtained depends on the specificity of the antibody used. These techniques can use fluorescence, peroxidase, etc., and do not require the presence of live pathogen.

All the cited pathogens are very likely amenable to the application of polymerase chain reaction (PCR) techniques to secure identification. However, for the time being this highly sensitive technique is almost entirely confined to research laboratories. In this context, PCR identifies the DNA of the different pathogens.

In the case of the indirect techniques the rationale is to search for antibodies that indicate that the pathogen has effectively infected the animal host. With practically all the pathogens cited, paired serological testing is of limited utility, since maternal titers are maximum at the time of abortion. However, in late abortive processes where the fetus does produce antibodies, the presence of the latter targeted to the pathogen constitutes an excellent diagnostic tool. On the other hand, it is important to take into account that with almost all vaccines the animals become seropositive.

Serology is indeed an excellent tool for eradicating disease (e.g., brucellosis), or for controlling the absence of infection in a certain group of animals (e.g., control of breeding animals).

Table 4. Habitual diagnostic techniques in application to reproductive problems.

After infection (vaccination), a primary IgM response is induced. These antibodies are pentavalent, and so are very well identified with agglutination techniques (Leptospira, Listeria), and their presence is indicative of recent infection. Such techniques offer only limited sensitivity in measuring the presence of IgG, however.

As can be seen in the figure, following the initial IgM response, IgG is produced. The IgG titer increases after the second exposure of the host; for this reason a variation in antibody titer (before and after abortion) can allow us to establish the underlying cause. However, this is not possible with only single sampling, since the presence of antibodies against a given process only indicates that the animals have been vaccinated, or that they have at some time come into contact with the disease.

Obviously, antibody studies allow us to know the prevalence of a disease. Table 5 provides some seroprevalence data corresponding to different pathologies. Seroprevalence informs us of the percentage of animals that have come into contact with the process, though such animals will not necessarily have aborted or suffered infertility alterations.

Table 5. Seroprevalence of the different etiologic agents.

Sampling and storage

It is advisable for the veterinarian to have access at least to the following material.

Material

Sterile tubes with heparin, shipment of blood samples for viremia searches (border disease carriers).

Airtight bottles to avoid leakage. Shipment of stools, exudates, milk, etc.

Syringes and sterile needles for collecting serum.

Airtight bags for submitting organs, tissues and fetuses. Sealing must be checked. Fine bags for freezing foodstuffs are quite acceptable, as they are resistant, thin, and can be knotted to ensure air tightness.

Swabs with transport medium (e.g., Amies or Stuart) for sampling in live animals, endocervical sampling and organ swab samples after necropsy.

White cork boxes for shipping refrigerated samples.

Ice-gel blocks for refrigerated samples. Frozen water should not be used.

Blood

Whole blood with anticoagulant. Refrigerated between +4 and +8ºC. The sample should reach the laboratory in under 24 hours after extraction. Protect the tubes against physical damage.

For lymphocyte culture (viremias): heparin.

Serum

In tubes without anticoagulant. No refrigeration is required if the samples are sure to reach the laboratory within 24 hours after collection. If storage for several days proves necessary (paired sera), the clean serum can be frozen after removing the clot. Never freeze sera with the clot, and protect the tubes against physical damage.

In order to maximize the volume of serum, after collecting the blood sample, allow the tube to stand at room temperature for about 30 minutes, in an inclined or inverted position.

Swabs or brushes

No refrigeration is required. The swabs are to contain transport medium (Amies or Stewart). Shipping to the laboratory should be made on the same day, though if storage is necessary, the samples should be refrigerated.

In live animals, swabs allow the collection of exudate with abundant cellular contents in the endocervical cavity. The swab should be introduced deeply within the cavity, rubbing against the walls while twisting or rotating the swab to collect the sample.

Fetuses

Fetuses require refrigeration. Do not freeze if bacteriological testing is requested. Freezing is only allowed if immunoperoxidase testing is required.

It should be remembered that contamination is likely if the dead tissues have been more than four hours without refrigeration. The samples must be packaged in an airtight container or bag.

Avoid the shipping of whole carcasses as far as possible — especially in summer. Small fetuses can be transported.

The samples should always be accompanied by:

Veterinarian / company data

Identification of the farm

Identification of the host species

Identification of the tissues submitted. In the case of swabs, indicate the zone from which the sample was collected.

A brief anamnesis.

Specification of the requested analysis or the clinical suspicion.

In the event refrigeration is needed, use gel blocks or dry ice. Do not use frozen water.

RESULTS OBTAINED IN OUR LABORATORY

Table 6. Origin of the samples received in our laboratory.

The distribution of the samples received in our laboratory corresponds strictly to the implantation of our center in the different autonomous communities in Spain, and perhaps to the different productive orientations of the farms. In this context, dairy farms invest more in diagnosis and prevention.

Table 7. Results obtained in our laboratory with immunocytochemical tests.

*SL: sudden loss; DIG: digestive; RES: respiratory; NER: nervous; OCU: ocular; ART: articular

The data reflected in Table 7 correspond to the results obtained in the cases received. In each case the laboratory may have received one or more fetuses, placentas, fetal tissues or endocervical swabs. In all cases studies were made of the pathogens requested by the veterinarian; consequently, the data shown cannot be taken to be indicative of prevalence.

Table 7 likewise provides some of the results obtained in other pathologies involving the same pathogen. In our opinion, these data can be useful, for they at least indicate the presence of such pathologies.

Of note from the data obtained is the important presence of Chlamydia (62%) and Campylobacter (51%). The low frequency of Salmonella spp. (5%) and the important role of Leptospira, Border and Toxoplasma are additional interesting findings. Listeria very likely plays a scantly important role in abortive processes, and Neospira probably lacks any importance at all in such situations — though a larger number of studies would be required to confirm this. Coxiella burnetti (Q-fever) has only recently been incorporated to our diagnostic battery, as a result of which the number of cases reported is not significant.

VALIDATION OF SWABS OF ORGAN SAMPLES

In diagnosing abortive processes, the fetus and/or placenta is always the best study sample. Obviously, the identification of a pathogen in its target organ is more informative than its identification in an endocervical swab. However, in many cases the fetus/placenta is not available, or is in poor conditions. In cases of infertility problems no fetus is available for obvious reasons. Finally, swabs allow the sampling of various animals — a fact that may increase the likeliness of identifying the presence of a pathogen.

Table 8. Results obtained with fetuses/placentas and endocervical swabs.

Two methods are available for contrasting the validness of immunocytochemistry in the diagnosis of abortive processes: comparison of the results obtained on receiving a swab and a fetus from the same animal, or comparison of the results obtained on studying an important number of cases.

Table 8 shows the results obtained with cervical swabs or with organs (fetuses, placenta, tissues, etc). As in other tables, a specification is made of the number of cases in which the cited pathogen has been identified, regardless of the number of samples received. More swabs than fetuses are normally received for performing the study, though in the case of fetuses we always work with two target organs when searching for pathogens.

As can be seen, no significant differences in the results are found, with the exception of Chlamydia. These results clearly reflect the reliability of using endocervical swabs in sampling. In the case of Coxiella, the number of samples compared is very small, and therefore lacks relevance.

As has already been mentioned above, in many of the cases received it is possible to identify the presence of more than one pathogen.

Based on 107 cases (investigating the presence of C. psittaci, T gondii, C. fetus and L. interrogans in all of them) in only 12 (11%) was no pathogen identified. In the 95 remaining cases the results were distributed as follows.

Table 9. Results obtained in 54 cases of simple infection.

Table 10. Results obtained in 41 cases of mixed infection.

In the same way as in the results obtained above, Chlamydia and Campylobacter continue to be the most prevalent pathogens.

The diagnosis of abortive processes is the responsibility of the clinical veterinarian — not of the diagnostic laboratory. In our opinion, the laboratory should make efforts to improve its analytical techniques, though it is finally the veterinarian who establishes the diagnosis.

In fact, the presence of a pathogen in a sample does not necessarily indicate the cause of abortion. Table 11 reports the presence of cases in which more than one pathogen is identified. The relative importance of these multiple pathogens in each case can effectively be appraised with the help of the laboratory (serologically confirming the antigenic findings, studying seroprevalences or titer changes, etc.), though under practical conditions this implies a substantial cost increment and delays in time.

The search for persistently infected (PI) animals

Asymptomatic carriers are infected during gestation, and thus do not recognize the border disease virus as foreign. For this reason these animals either produce no antibodies or produce very little immunoglobulin targeted to the virus.

In order to identify PI status in an animal, the first step is to search for individuals who do not produce antibodies.

1.- Antibody detection

In inactivated vaccines the p80 protein is lost; consequently, if antibodies against p80 are present, the animal is diagnosed with field virus infection.

In an ELISA block assay, the test serum antibodies inhibit adherence of the antibodies supplied with the kit (added in a second phase).

The distribution of results is as shown in the figure. Positivity is considered for those animals with sufficient antibody production to block adherence of 50% of the antibodies of the kit, while negativity corresponds to those cases that are unable to block even 30%. In turn, doubt concerning PI status is defined by inhibition rates of 30-50%.

In order to improve the reliability of identifying carrier status, we select all animals exhibiting a percentage block of under 54%, i.e., all the negative cases, all the doubtful cases and also the weakly positive individuals.

2.- Antigen detection

The PI animal is viremic; consequently, to identify such individuals it is necessary to detect the blood circulating virus. Antigen-capture ELISA kits are available; however, as with other ELISA techniques, they produce a discouraging range of doubtful cases. The alternative here is to perform in vitro cultures of the blood cells.

The mononuclear cells in heparinized whole blood are separated on a density gradient, followed by counting and culture for 24 hours in the presence of a stimulant. In this way the virus responds to activation of the cells by multiplying and expressing its antigens.

The cells extracted from culture are washed several times, fixed on slides and labeled with monoclonal antibody against proteins p80/p125. In all viremic animals this study should be repeated after 40 days. Approximately 5-7% of the animals are viremic in the first study, though only 1-2% are viremic in the second.

Control of breeding animals. Semen control

Based on the above considerations, it is of interest to focus on the health of breeding animals, particularly those dedicated to semen production for artificial insemination purposes (AI).

In general, the non-vaccinated selection males can be adequately controlled for health by serological testing. However, in the case of vaccinated animals it is not possible to distinguish between vaccinal and disease-induced antibodies. In such situations it is possible to search for pathogens in semen, e.g., C. psittaci or Campylobacter spp.

In this context, it is worth drawing attention to some of the specific germs responsible for problems of epididymitis, orchitis, etc.: Actinobacillus seminis, Arcarobacterium pyogenes, Staphylococcus aureus, etc. Such pathogens can also be identified by microbiological analysis. For these cases it is necessary to remember that semen sampling is complicated by the risk of contamination (to be cleaned with appropriate detergents), and that some of these pathogens are strict anaerobes.

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