Chlamydia abortus

Causative agent of Ovine Enzootic Abortion (OEA), a disease that is widely distributed worldwide, which causes economic losses in countries that are engaged in livestock activity. The disease causes abortion in pregnant ewes in the last third of gestation or, in some cases, the birth of weak lambs that do not exceed 48 h of life^1,2. It is currently considered the most important pathology of chlamydial origin; since it represents a potential occupational zoonotic risk and for pregnant women who are in contact with infected animals². In this order, different reports have been made in which, in addition to causing abortions, it causes other conditions, such as: febrile illness, development of disseminated intravascular coagulation, acute renal failure and pulmonary edema¹⁰, septicemia and important lesions in the liver, kidney and heart; it is worth mentioning that these pathologies occurred after the abortion¹¹. It is also described as a causative agent of pelvic inflammatory disease¹². In Mexico, the prevalence of antibodies against C. abortus was reported in exposed risk groups (workers and veterinarians) who were in contact with flocks with a history of abortion¹³. Finally, it has also been reported as a causative agent of pneumonia problems¹⁴, thus demonstrating the zoonotic potential of this bacterial species.

Chlamydia psittaci

Bacterium of avian origin that causes psittacosis in birds and with proven potential zoonotic risk. In the last five years, different studies have been carried out, evidencing the risk that this bacterial species represents for humans, mainly due to contact with infected birds². In the first instance causing psittacosis, ornithosis² or atypical pneumonia¹⁵. It has also been reported as a causative agent of genital infections in women¹⁶. It has been identified in patients with respiratory diseases; for example, pneumonia in farmers who worked with infected animals¹⁷. Similarly, it has been linked to pneumonia, pertussis and conjunctivitis¹⁸. Not least, associating the possible risk factors involved in the contagion of psittacosis in people who handle birds^19,20. Recently, it was reported as a causative agent in an outbreak related to severe respiratory disease among workers of poultry slaughter plants in the USA²¹. There are currently no reports of contagion of C. psittaci from mammals to humans.

Chlamydia pecorum

This bacterium lodges naturally in the digestive tract and causes enteritis; in most cases, it occurs subclinically, thus avoiding the timely detection of the disease²². C. pecorum has also been associated with other diseases, among them: arthritis, keratoconjunctivitis, encephalomyelitis, infertility, pneumonia and mastitis, and causing economic losses in production units²². Despite the wide variety of pathologies with which this bacterial agent has been related, the zoonotic potential of this species of Chlamydia is still unknown²³.

Enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) is an indirect diagnostic test that detects antibodies in serum of affected individuals against specific antibodies of bacteria of this genus^2,24, there are currently commercially available ELISA tests, among the disadvantages of this type of tests are cross-reactions between species (C. abortus and C. pecorum), which makes specific diagnosis difficult². The assays that evaluate different antigens for the detection of species of Chlamydia spp. have been diverse. First, evaluating fragments of the MOMP through an indirect ELISA test (rOMP91B iELISA), which showed a sensitivity and specificity of 84.2 and 98.5 % respectively; the study showed that the indirect ELISA test was better at differentiating animals infected with C. abortus and C. pecorum²⁶. Later, another study evaluated different recombinant antigens, all of these were identified from the Polymorphic Outer Membrane Protein or POMP90. Of the 11 fractions identified, OMP90-3 and OMP90-4 were the most effective, showing a sensitivity of 95.7 and 94.3 % respectively and a specificity of 100 % for both. The findings of the study revealed that the ELISA test with the fragment rOMP90-4 was more sensitive than that of rOMP90-3, since it identified more positive samples for OEA and, in addition, both were superior to the complement fixation test (CFT)²⁷. Additionally, and in a complementary way to these studies, a study was carried out in which four experimental ELISA tests based on complete EB of C. abortus (EB), a preparation from the outer membrane of complete bacteria (SolPr) and two recombinant fragments of POMP90 (rOMP90-3 and rOMP90-4), were evaluated against three commercial tests, the CHEKIT1 Chlamydophila abortus test, Pourquier1 ELISA Chlamydophila abortus and ImmunoComb Ovine Chlamydophila Antibody. The results during the test showed that the commercial ELISA test InmunoComb obtained the highest sensitivity (98.4 %) compared to the others; however, the specificity determined (65.4 %) was lower than all tests evaluated. The results at the end of the study determined that, of the eight ELISA tests evaluated, the test that offers the best results in terms of sensitivity and specificity was the ELISA test based on the recombinant fragment rOMP90-3, with values of 96.8 % and 100 % respectively, this study showed that this experimental ELISA test can be an adequate alternative for the serological diagnosis of OEA²⁸.

A study carried out in 2018 adds to these results, which compared three commercial tests (IDvet, MVD-Enfer and LSI) for the detection of antibodies against C. abortus in ewes, vaccinated animals were evaluated for different periods of time to measure the production of antibodies between animals that aborted and animals that reached term, the results revealed that the most sensitive test was the LSI (94.74 %) followed by MVD-Enfer (78.95 %) and finally IDvet (73.68 %), all three kits detected high levels of antibodies in ewes that aborted compared to those that had lambs without complications. The most sensitive test in this study is based on the identification of chlamydial lipopolysaccharide (LPS), which shows cross-reaction with all species of the genus Chlamydia, it is determined suitable for identifying ewes infected with any species of Chlamydia, but it is not considered specific for C. abortus²⁹. Finally, a study revealed the disadvantages of this type of commercial tests in terms of cross-reactions between C. abortus and C. pecorum, carrying out the evaluation in different flocks, serological tests revealed a low seropositivity of C. abortus using an ELISA test based on peptides (1.2 %) in Australian ewes and a moderate seropositivity in a Swiss flock with a clinical history of abortion associated with C. abortus (26.9 %). Using CFT and ELISA tests, seropositivity was significantly higher, suggesting cross-reactivity between these two species. Additionally, using a real-time PCR test to detect DNA of C. pecorum in Australian animals seropositive for Chlamydia spp., it was concluded that the seropositivity of Chlamydia may be related to cross-reactivity with endemic infections of C. pecorum³⁰. Due to the disadvantages shown by this type of tests, it is advisable to complement them with a more specific one such as PCR tests²⁴.

Cell culture

Due to their obligate intracellular nature, these bacteria require live media for their isolation; due to this, cell culture (McCoy cells by choice) is currently used for this purpose²⁴, until a few years ago, this technique was considered the gold standard for the diagnosis of Chlamydia spp.¹; however, the development of new methodologies, such as nucleic acid amplification (PCR and sequencing), employed to improve diagnosis are currently considered the gold standard for diagnosing Chlamydia spp. infections⁸. Other tests such as polymerase chain reaction, which is a much more specific technique for the detection and typing of species of Chlamydia spp., is used much more frequently because it has a greater sensitivity and specificity compared to cell culture².

Polymerase chain reaction test

This test detects specific DNA of any organism; therefore, it is a much more sensitive and specific test than cell culture when identifying the genus and species involved in the affected individuals²⁴. In the last 15 yr, the use of this technique has become very relevant, and its different variants reveal better results compared to those mentioned above, such as: i) processing a greater number of samples, ii) less time to obtain results, iii) the use of different types of samples for diagnosis, iv) organisms do not have to be one hundred percent viable and v) greater sensitivity and specificity. Since its use in veterinary diagnostic laboratories was implemented, the genes used to identify these bacterial agents have been different: major outer membrane protein (MOMP), polymorphic membrane proteins (Pmps), 16S and 23S²⁴. PCR methodologies for the specific detection of Chlamydia species are varied, to mention a few: The PCR test “Touchdown enzyme time release” to amplify different DNA sequences in the variable regions of the spacer rRNA genes 16S and 16S-23S specific for the identification of species of this bacterial genus; for example, Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci³¹. Another variant of this test, the PCR-RFLP test that identifies in the first instance the presence of the omp2 gene specific to the family Chlamydiaceae and later, by digestion with the restriction enzyme AluI, has the ability to identify a total of nine species of the genus Chlamydia, among these, C. abortus, C. pecorum³² and C. psittaci³³. Additionally, a variant focused on the 16S gene specific to the family Chlamydiaceae but using a real-time PCR test demonstrated high specificity when evaluating samples of different Chlamydia species against other bacterial genera³⁴; additionally, this variant can also be used for the specific identification of Chlamydia species using specific primers for each one³⁵. Later, a multiplex PCR test was developed for the identification of C. abortus, C. pecorum and Coxiella burnetii, involved as causative agents of abortion, this test, unlike the aforementioned ones, helps to simultaneously identify the three species, this test proved to be highly specific and rapid for the detection of these bacterial agents³⁶.

Antibiotics

For the treatment of pathologies of chlamydial origin, antibiotics are the drugs of choice, since bacterins, in the case of OEA, are only available in some European countries and the costs required for their implementation are very high^2,7. The administration of tetracycline, penicillin and chloramphenicol for the treatment of infections caused by these bacterial agents has been shown to inhibit the growth of these organisms²⁴, it is important to emphasize that the use of antibiotics should be in a controlled manner to minimize the development of resistance by the pathogen. Although antibiotics serve to reduce losses due to these pathogens, these types of treatments do not eliminate bacteria; since affected animals continue to shed the organisms; therefore, their prophylactic use is not recommended³⁷. In European countries, in addition to the use of antibiotics, the administration of bacterins is also implemented in the case of OEA to prevent and control the disease in flocks with susceptible animals, this is because it is the only disease of chlamydial origin for which there are commercially available bacterins².

Immunogens

In the past century, immunogens intended for the treatment and prevention of diseases worldwide have been one of the greatest achievements of public health; in this area, between 2 and 3 million lives are saved due to the implementation of these health measures³⁸. In the veterinary field, technological advances in the development of immunogens for disease control have had an important enhancement in the last 25 yr, from the use of complete bacteria, whether alive or dead, to the use of DNA immunogens, which offer safer measures for both the animal and the veterinarian who administers them³⁹.

In the last 70 yr, studies for the development of immunogens to combat diseases of chlamydial origin in animal species, mainly livestock (sheep, goats, cattle and pigs), focus on preventing economic losses in production units; however, the main objective is to preserve human health due to the zoonoses that some of these represent. Vaccine trials against Chlamydia spp. have increased in the last 10 yr, in these studies, the protein most used in the challenges against Chlamydia spp. has been the MOMP⁴⁰. Additionally, studies focused on the search for specific antigens of this surface protein have been carried out for a differentiation between species⁴¹. Later, different types of antigens have been used in the development of immunogens against chlamydial agents, the first tests traditionally used the EB, which were inactivated by treatments with ultraviolet light or live (attenuated) fixed with formalin. Later, in the mid-1990s, approaches in the use of other antigens for the development of subunit vaccines, such as recombinant proteins, synthetic peptides, expression vectors and DNA, began to be used for the challenge in murine models mainly⁴⁰.

In the case of C. pecorum, there are only two trials of vaccines against this agent, which have been challenged in murine models, although the results revealed an immune response in animals, these should be treated with caution because clinical cases of abortions are rarely related to C. pecorum⁴².

Finally, although there are commercially available vaccines for the control and prevention of C. abortus, it is well documented that in the case of the live attenuated 1B C. abortus vaccine, it has the potential to reactivate and cause the disease in immunized animals^43-45.

Of the total vaccine challenges against Chlamydia spp., only 5 % have been focused on C. abortus, evaluated in mice, cows, ewes and guinea pigs⁴⁰, the studies evaluate some proteins, mainly Pmps, looking for different variations or mutations that can serve as key points for the prevention of OEA⁴⁶.