Chlamydial Infections in Dairy Cows & Vaccination & Final Discussion
ACVIM 2008
Bernhard Kaltenboeck, Dr. med. vet., PhD
Auburn, AL, USA


Historically, the existence of latent chlamydial infections in livestock had been well recognized, but their impact was not understood because of the difficulty detecting these infections and the resulting uncertainty about the overall prevalence of chlamydial infections in animals.1 Improved diagnostics, both by serology and PCR-detection of chlamydial DNA, have changed the historical perception that detectable chlamydial infection in animals is typically associated with clinically apparent, frequently severe disease, such as enteritis and pneumonia in birds with ornithosis, or abortion, conjunctivitis, polyarthritis, or encephalomyelitis in livestock. Veterinary chlamydiologists are now faced with the conundrum of a high frequency of detectable chlamydial infections, but a dearth of significantly associated clinical disease manifestations.2-5

Demonstration of health effects of these widespread, low-level endemic chlamydial infections represents a challenge, but also a great opportunity for a contribution to an improved understanding of such infections that similarly affect humans, such as in the association of C. pneumoniae with atherosclerotic lesions. Health effects of subclinical infections may have a profound impact on livestock productivity and farm income, if these infections affect the respiratory and intestinal tract of young animals in which they may reduce growth rates, or the reproductive organs of adult animals in which they may affect fertility or milk production. The classic concept of prophylactic immunization that elicits sterilizing immunity and virtually 100% protection from disease does not apply to chlamydiae. However, accumulating evidence indicates that therapeutic vaccination may nevertheless provide substantial health and economic benefits. By influencing the outcome of chlamydial infections, therapeutic vaccination may also provide an intervention approach that helps to elucidate the full health and economic impact of clinically asymptomatic chlamydial infections in livestock. Some of the most interesting advances in veterinary chlamydiology come from "production medicine" studies that address the subtle health effects of these chlamydial infections.

Prophylactic Vaccines Against Animal Chlamydioses

Generally accepted animal chlamydial diseases that call for prophylactic vaccination are presently abortion in small ruminants, the respiratory disease complex of turkeys in which C. psittaci infection is an important component, and conjunctivitis and respiratory infection in cats. While both attenuated live and inactivated vaccines against C. felis are in use for pet cats, little has been reported about their efficacy. Vanrompay et al. used genetic immunization to vaccinate turkeys with C. psittaci ompA, and achieved significant reduction in chlamydial shedding, and virtually complete protection from disease after respiratory challenge with C. psittaci.6,7 A live vaccine of an attenuated, temperature-sensitive strain of C. abortus (Enzovax®, Intervet) reportedly reduces chlamydial abortion in small ruminants.8 However, convincing challenge and epidemiological efficacy studies of any prophylactic vaccine are not available, none mediates sterilizing immunity, and the prophylactic efficacy presumably is low.

Components for a Viable Subunit Vaccine

While live and inactivated C. abortus vaccines for prevention of sheep/goat abortion are available, there is nevertheless considerable interest in improved vaccines of higher efficacy against chlamydial infections in livestock that also allow discrimination between vaccinated and naturally infected animals. Therefore, future viable chlamydial will have to be synthetic subunit vaccines, composed of few, optimally suited chlamydial antigens and adjuvants. Another reason for subunit vaccines is that certain components of whole chlamydiae presumably are critical mediators of chlamydial disease; and finally, production of chlamydial elementary bodies for whole-organism vaccines is expensive and inefficient. Therefore, a rational search for antigens and adjuvants is required.

Vaccine Candidate Antigens

Several groups have used models of mouse infection with C. abortus or of the original host infection with C. psittaci to identify vaccine candidate antigens or to test delivery modalities of vaccines. Héchard et al. tested the protective efficacy of the C. abortus ompA and groEL genes by genetic immunization, but did not obtain significant reduction in chlamydial loads or protection of fetuses after intraperitoneal challenge inoculation of pregnant vaccinated mice with C. abortus.9,10

For potential use in a future vaccine against C. abortus, Stemke-Hale et al. used expression library immunization (ELI) of mice with pools of plasmids, starting with 80,000 random inserts of the C. abortus genome, for identification of protective vaccine candidate genes.10 ELI is an unbiased whole-genome approach in which pools of expression plasmids containing inserts from the C. abortus genome were tested in a mouse pneumonia model for the ability to confer protection against challenge infection. Pools of protective plasmids were fractionated into smaller pools, until single protective gene candidates were tested. Five gene fragments conferred protection at levels as good or better than the live-vaccine control of mice that had received a low-level respiratory C. abortus inoculation one month prior to the high-dose challenge inoculation. Four of these fragments (dnaX2, gatA, gatC, pbp3) encoded peptides of cytosolic proteins; only one (pmp5) encoded a peptide of an outer membrane protein, traditionally considered the best vaccine candidates. The dnaX2 fragment, encoding a portion of the C. abortus DNA polymerase, completely protected mice from C. abortus disease, better than the best protection achieved with live vaccination by low-level intranasal C. abortus inoculation.10

Vaccine Adjuvant

In a systematic approach, Caro et al. examined the influence of the inactivation method of C. abortus elementary bodies and of the type of adjuvant on the protective efficacy of a killed vaccine against intraperitoneal challenge with C. abortus.11 They found that best reduction in chlamydial organism load on day 4 after inoculation was mediated by a killed vaccine prepared by inactivation of chlamydial organisms by binary ethylenimine and adjuvanted with QS-21, a purified Quillaja saponaria saponin, or with Montanide 773. These data may be helpful in formulating future subunit vaccines.

Clinically Asymptomatic Chlamydial Infections in Dairy Cows

The ability of chlamydiae to chronically infect virtually any organ system, combined with their ubiquitous distribution, makes chlamydiae prime candidates for involvement in costly, low-level infections of organs of particular importance in production medicine. Diseases of reproductive organs in dairy cows affect fertility as well as milk production, and fertility disorders and mastitis are among the most important livestock diseases. Chlamydiae have been conclusively identified as causal factors in these diseases. Several reasons make a vaccine against these bovine chlamydial infections desirable: 1) it is likely that vaccination can create a differential in immunity to C. abortus that would aid to evaluate the impact of subclinical chlamydial infection on bovine fertility in controlled trials; 2) prophylaxis or therapy of chlamydial infections by vaccination is highly preferable in production animals as compared to regimens using antibiotics; 3) depending on future findings on the impact of chlamydial infection in beef and dairy cattle, successful vaccination might substantially improve bovine herd health.

Bovine Fertility

DeGraves et al. investigated the effects of controlled re-infection on the fertility of cattle naturally pre-exposed to C. abortus.13 All animals had high pre-challenge levels of IgM, IgG, IgG1, and IgG2 serum antibodies against ruminant C. abortus in a chemiluminescent ELISA. Twenty virgin heifers were estrus synchronized with prostaglandin F2, artificially inseminated 2-3 days later, and challenged immediately by intra-uterine administration of 0, 104, 105, 106, or 108 inclusion forming units (IFU) of C. abortus. Ten heifers were estrus-synchronized, inseminated, and uterine-challenged 2 weeks later. These animals were also indirectly exposed to C. abortus infection (cohort challenged) by contact with their previously challenged cohorts. Pregnancy was determined by rectal palpation 42 days after insemination. No animal showed signs of clinical disease. One hundred percent, 83%, 50%, 66%, and 0% of heifers were pregnant after uterine challenge with 0, 104, 105, 106, or 108 IFU of C. abortus, respectively. Fifty percent and 65% of heifers were pregnant with or without cohort challenge, respectively. Uterine inoculum dose and cohort challenge, or alternatively a negative pregnancy outcome (infertility), correlated highly significantly with a rise in post-challenge over pre-challenge anti-C. abortus IgM. Logistic regression significantly modeled that the uterine C. abortus inoculum causing infertility is 8.5-fold higher for heifers without cohort exposure and 17-fold higher for heifers with high IgM than for heifers with cohort exposure or with low IgM. This investigation demonstrated that an asymptomatic, circulating, non-sexually transmitted herd infection by C. abortus has a profound negative influence on the fertility of cattle bred at this time.14

The protective genes identified by Stemke-Hale et al. and their corresponding proteins were used in a preliminary vaccine trial to evaluate protection against C. abortus.15 The five most protective genes were selected for vaccination of virgin heifers in a C. abortus cohort challenge model. This vaccine was composed of a pool of plasmids that contained the full ORFs of the genes, recoded for mammalian codon usage. A corresponding recombinant protein vaccine against C. abortus was also developed. This vaccine was Alum-Quil A based and contained affinity-purified full-length proteins expressed in E. coli, using the same genes that were incorporated into the genetic vaccine.

A natural Holstein heifer infection model was used in which intracervically inoculated--but not bred--heifers transmitted C. abortus to, and induced infertility in, herd mates that were bred 2 weeks later and were indirectly C. abortus challenged by contact exposure (cohort challenged). This model built on the experience obtained in the uterine C. abortus heifer challenge experiment outlined earlier. The experimental herd mates were used to evaluate the genetic and recombinant protein vaccines, compared to mock vaccinated controls. Twenty-seven heifers were synchronized for estrus and then inoculated with an intrauterine dose of C. abortus. When the challenged heifers were at maximum shedding of C. abortus, estrus was synchronized in experimental heifers (n=24). Experimental heifers were then monitored for estrus with a computerized estrus detection system and bred by artificial insemination. Pregnancy was determined by rectal palpation at 42 days post breeding. The results shown in Table 1 suggest protection by both genetic and protein vaccines from C. abortus-induced infertility. The odds ratio for improvement of fertility with vaccination against C. abortus is 4.5 (p=0.12).

Table 1. Fertility of vaccinated heifers 42 days after breeding and cohort challenge with C. abortus15.

Vaccine Group


Not Pregnant


6 (50%)

6 (50%)


4 (80%)

1 (20%)


5 (83%)

1 (17%)

The ~32% increase in fertility of vaccinated heifers as compared to the 50% first-service conception rate of control heifers would represent a substantial improvement in bovine herd health. Thus, immunization with a subunit C. abortus vaccine may hold promise to improve bovine herd fertility. It is important to note that the vaccines did not create a fresh adaptive immune response to C. abortus in naive heifers, but rather enhanced and modulated an existing response in animals that partially had detectable chlamydial infection. Thus, the vaccines were used as therapeutic, not prophylactic vaccines, and may be termed "antigen-specific immune modulators".

Chlamydial Infection of the Bovine Mammary Gland

Another approach at analyzing the effect of clinically inapparent chlamydial infections in cattle was chosen by Biesenkamp-Uhe et al.16 Mastitis is the economically most important disease in animal agriculture, affecting both milk quantity and quality. Most cases of mastitis in dairy cattle are clinically inapparent, and typical mastitis pathogens such as Streptococcus agalactiae are detected only in a fraction of the cases. Subclinical mastitis is nevertheless of major interest to production medicine because of the large impact on profit margins of dairy farms. Infections with C. abortus and C. pecorum are ubiquitous in cattle, and have been experimentally and clinically associated with bovine mastitis. In a prospective cohort study in a herd of 140 Holstein dairy cows they examined the influence of chlamydial infection detected by PCR on subclinical inflammation of the bovine mammary gland as characterized by elevated somatic cell counts (SCC) in milk. SCCs are a sensitive quantitative indicator of inflammation, and 105 somatic cells per ml milk are considered the upper limit for a healthy bovine mammary gland. All cows had serum antibodies against Chlamydia, and 49% of the cows were positive for C. abortus on day 0 on at least one PCR of a conjunctival or vaginal swab from day 0 of the experiment. Chlamydia infection and below-median anti-chlamydial serum antibody levels significantly associated with bovine subclinical mastitis in this investigation.16 An intervention approach by perturbation of the immune response to C. abortus/C.pecorum was used to further examine induction, and immune-mediated reduction, of mastitis caused by chlamydial infection. All dairy cows had established immunity to chlamydiae, and serologically- and/or PCR-demonstrated chlamydial infection. They received two doses of an inactivated Alum-Quil-A-based vaccine of C. abortus/C. pecorum elementary bodies (therapeutic vaccination) or a mock vaccine on days 0 and 35 of the investigation. This vaccination highly significantly reduced milk SCC (Figure 1), thus reduced bovine mastitis, and increased anti-chlamydial antibody levels, but did not reduce shedding of Chlamydia bacteria. Chlamydia vaccination also resulted in improved relative body condition of dairy cows after 10 weeks. The disease-protective effect was maximal 10 weeks after vaccination, and lasted for additional 4 weeks. Vaccination with the C. abortus/C. pecorum vaccine, the mock-vaccine, or an unrelated vaccine against Bovine Viral Diarrhea virus resulted in highly significant transient increase in chlamydial shedding in milk, presumably mediated by the vaccine adjuvant. This investigation demonstrated an etiological involvement of the ubiquitous chlamydial infections in bovine mastitis, a herd disease of critical importance for the dairy industry. Furthermore, it shows the potential for transient improvement of chlamydial disease by therapeutic vaccination.

Figure 1.
Figure 1.

Effect of Chlamydia vaccination on milk somatic cell counts (SCC).16

Chlamydia vaccine, o mock vaccine, data are normalized to identical day-0 means of Chlamydia- and mock-vaccinated animals; means ± 95% CI. Chlamydia-vaccinated cows have significantly lower milk SCC than mock-vaccinated cows for all combined time points after day 0, (p=0.007).16


The ever increasing data from epidemiological surveys, both of domestic and of feral animals, indicate that chlamydial infection of animals, by classical Chlamydiaceae species, is the rule rather than the exception. PCR detection typically indicates low numbers of the organisms, and the vast majority of these infections are without obvious clinical symptoms, suggesting overall mostly endemic infections. The dominant maintenance mechanism of chlamydial infections in the host populations appears to be frequent and clinically inapparent re- or superinfection coupled with the slow elimination of these agents by host immunity. Only if several epidemiological risk factors coincide, such as stress imposed on a susceptible, high-density host population, do these infections build up to become clinically manifest. Emerging data, though, indicate that the inapparent infections are not innocuous, but do cause minor inflammatory reactions and increase susceptibility to viral and bacterial superinfection. While clinically manifest chlamydial diseases are rare and affect only a small fraction of, or infrequently the whole, host population, inapparent infections affect in subtle ways every member of the population. For that reason, subclinical chlamydial infections are probably economically more important in livestock than classical chlamydial diseases. The future challenge for veterinary chlamydiology will be to dissect the impact of the endemic animal chlamydial infections, and devise strategies to ameliorate their negative effects. Vaccines against animal chlamydiae have the potential to be used as perturbation tools in such studies, as well as instruments for control of animal chlamydial infections, potentially substituting for presently widely used antibiotics.


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Speaker Information
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Bernhard Kaltenboeck, DMV, PhD
Auburn University
Auburn, AL

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