Prevalence of chlamydiae in dairy cattle herds and factors contributing to the spread of infections

Chlamydiae are Gram-negative obligate intracellular bacteria belonging to the Chlamydiaceae family. Chlamydia spp. are widely distributed throughout the world and cause a variety of diseases both in humans and animals, including zoonotic infections (8). Currently, 15 Chlamydia species are recognised and four are Candidatus species (31). The most common chlamydial agents in cattle are C. pecorum and C. abortus. However, because of their ability to cross natural host barriers, C. suis (endemic in swine) and C. gallinacea (endemic in chickens) have sporadically been recorded as causing asymptomatic infections in cattle, while C. psittaci (of which birds are the reservoir) has also been noted to cause an infection associated with clinical signs (12, 13). Moreover C. abortus co-infections with other chlamydiae species are also possible.

Chlamydiosis is a multifactorial disease that involves interactions among low-virulence pathogens, nutritional deficiencies, poor management and hygiene and host genetics (17). Chlamydia abortus and C. pecorum affect cattle, causing abortions and reproductive problems that may lead to significant economic losses. The disease has a nonspecific clinical presentation, and clinical symptoms such as pneumonia, conjunctivitis, enteritis, polyarthritis, sporadic encephalomyelitis, abortion, vaginitis, endometritis, repeat breeding, weak calf syndrome, perinatal mortality and fertility disorders have been reported around the world (13). Infection with Chlamydia spp. could also have a subclinical course. Chlamydia abortus has been identified in suboptimal production of livestock in the absence of other pathogens (21). It is commonly known as zoonotic agent (21), and pregnant women may develop life-threatening C. abortus infections, resulting in septic abortion and stillbirth (27). For a long time C. pecorum was considered non-pathogenic to humans, but recently the first case in a man with pneumonia and respiratory failure was reported (4).

Taking into account the zoonotic risk and negative impact of chlamydiae on breeding cattle, surveys monitoring their prevalence are essential. Relevant data applying to Polish cattle herds are very limited. The studies conducted in the last decades showed that chlamydial infections are present in Polish bovine herds (16, 25), but current epizootic data from Poland are lacking.

Coxiella burnetii is the second intracellular and zoonotic agent that commonly occurs in the dairy cattle population worldwide, including Poland (24). Although Coxiella burnetii and chlamydiae belong to phylogenetically unrelated species, they show some similarities in their interaction with the host and pathogenesis of the infection (14). The clinical features of abortion caused by Chlamydia spp. and Coxiella burnetii are very similar, and such mixed infections have been suggested to be a common occurrence in sheep and goats (1).

The aim of this study was to establish the prevalence of chlamydiae in dairy cattle herds in Poland based on analyses of sera by the complement fixation test (CFT). It also estimated the true prevalence (TP) to chlamydiae at the animal and herd levels in individual Polish voivodeships using appropriate statistical methods. The investigation also evaluated the shedding of the pathogen by molecular testing of vaginal swabs and placentas collected from seropositive animals. Lastly, it statistically evaluated the conformity between seroprevalence of Chlamydia spp. and Coxiella burnetii in sera.

Sera samples were collected between 2019 and 2023 in the ambit of the “Protection of animal and public health” multiannual monitoring programme. Depending on which material was available, swabs and placentas were obtained from seropositive cows. All samples were collected from random animals by authorised veterinarians (veterinary inspectorate employees), following standard procedures and with farmers’ consent. According to the Local Ethical Committee on Animal Testing at the University of Life Sciences in Lublin (Poland), formal ethical approval is not required for this kind of study.

Serological analyses of sera samples confirmed the presence of antibodies against chlamydiae in 866 out of 2,780 (31.2%) tested animals with a 95% confidence interval (CI) of 29.4–32.9%. The overall herd-level prevalence was 42.7% (492/1,153) with a 95 % CI of 39.8–45.6%. Positive cattle herds were present in all tested voivodeships and seropositivity varied from 23.7% (CI: 17.6–30.7%) to 80.0% (44.4–97.5%) among them. Lesser Poland and Greater Poland were the provinces with the highest percentage of seropositive cattle herds (80%; CI: 44.4–97.5% and 73.7%; CI: 48.8–90.9%, respectively), while the lowest herd prevalence was observed in Subcarpathia (23.7%; CI: 17.6–30.7%). The highest percentages of positive cows were noted in Świętokrzyskie (38.3%; CI: 29.6–47.6%), Greater Poland (37%; CI: 29.2–45.4%), Podlaskie (36.6%; CI: 29.4–44.2%), Łódź (36.2%; CI: 29.5–43.4%) and Kuyavia-Pomerania (36%; CI: 30.0–42.3%) provinces. The lowest seropositivity rates of tested specimens were calculated for Warmia-Masuria (23.3%; CI: 16.8–30.9%), Subcarpathia (24.2%; CI: 18.1–31.1%) and Lesser Poland (25.6%; CI: 16.9–35.8%). The results of the serological testing of sera, including the overall prevalence and the true prevalence estimates, are shown in Table 1.

Next, the TP was estimated using the Bayesian framework. The posterior median and 95% confidence intervals were displayed. By including the voivodeship as a covariate in the model, it was possible to estimate the seroprevalence of Chlamydia spp. per voivodeship. A large variation in seroprevalence at the animal and herd levels between the voivodeships was observed. The TP values at herd level were the same as for apparent prevalence, but the Clopper–Pearson 95% CIs were different. The mean TP at animal level was slightly higher and was 33.3%, with a CI of 21.2–76.8%, individual voivodeship TPs ranging from 23.9% (CI: 8.5–63.0%) to 43.3% (CI: 28.3–95.9%). The highest values of TP at herd level were noted in the Lesser Poland voivodeship at 80.0% (CI: 49.7–95.6%), Greater Poland at 73.7% (CI: 51.6–89.2%), and West Pomerania at 67.4% (CI: 52.7–80.0%), whereas the highest values of TP at animal level were seen in the Świętokrzyskie voivodeship (43.3%; CI: 28.3–95.9%), Łódź (41%; CI: 27.0–95.1%) and Greater Poland (40.3%; CI: 26.9–95.5%). The lowest values of TP at herd level were noted in Subcarpathia (23.7%; CI: 17.8–30.6%), and at animal level in Warmia-Masuria (23.9%; CI: 8.5–63.0%). The true prevalence at the animal and herd levels in voivodeships is presented on the map in Fig. 1.

Fig 1.

True seroprevalence of chlamydiae at animal and herd level in cattle herds in the voivodeships of Poland based on serum analyses by complement fixation test. TPH – true-positive herds; TPA – true-positive animals; Voivodeships: GP – Greater Poland; PDL – Podlaskie; SW – Świętokrzyskie, LDZ – Łódź; KP – Kuyavia-Pomerania; MAS – Masovia; OPO – Opole; LP – Lesser Poland; SUB – Subcarpathia; WP – West Pomerania; LB – Lublin; SL – Silesia; POM – Pomerania; LS – Lower Silesia; LU – Lubusz; WM – Warmia-Masuria

– the size of the symbol corresponds to the number of cows in the voivodeship

Statistically significant differences were found between the lowest TP value at herd level noted in the Subcarpathia voivodeship and the equivalents in the Kuyavia-Pomerania, Warmia-Masuria, Lesser Poland, Masovia, Opole, Podlaskie, Pomerania, Świętokrzyskie, Greater Poland and West Pomerania voivodeships. A statistically significant difference was also observed between the highest TP value recorded at herd level in the Lesser Poland voivodeship and same statistics in the Lublin, Łódź, Opole, Greater Poland and West Pomerania voivodeships. Significant differences were also noted between the herd-level average TP value recorded in the West Pomerania voivodeship and these values in the Warmia-Masuria, Silesia, and Kuyavia-Pomerania voivodeships. There were no significant differences in average TP at animal level across the various voivodeships, which is related to the high uncertainty of TP resulting from the poor diagnostic parameters of the CFT method.

The results of MVA statistical analysis include the following factors: the presence of antibodies against Chlamydia spp. in sera at herd and animal level, the geographical location of the farms and the dairy cow stock in the voivodeship and positive and negative conformity rates between results of serological screening for Chlamydia spp. and Coxiella burnetii in the same sera samples. The analysis identified five groups of voivodeships with noteworthy differences (Fig. 2).

Fig. 2.

Multivariate analysis of the presence of antibodies against chlamydiae in sera at herd and animal level, the geographical location of the farms and the dairy cow stock in the voivodeship and the positive and negative conformity rates between the results of serological screening for chlamydiae and Coxiella burnetii in the same sera samples. Blue points represent each category of analysed variables. Blue points with similar profiles (low value of distances indicating a strong association between variables) are marked by the red rectangles. Blue points located in the green rectangle in the graph’s centre show the points with similar profiles but representing eigenvalues indicating the lack of any association. Blue points located in the dashed rectangle shows the points with similar profiles but representing eigenvalues indicating the association with more than one groups

ACh+ – seroprevalence of chlamydiae at animal level; HCh+ – seroprevalence of chlamydiae at herd level; CP – dairy cattle population; Con Pos Q/Ch – conformity between positive results of presence of Chlamydia spp. and Coxiella burnetii antibodies in the same sera samples; Con Neg Q/Ch – conformity between negative results of presence of Chlamydia spp. and Coxiella burnetii antibodies in the same sera samples. Voivodeships: GP – Greater Poland; PDL – Podlaskie; SW – Świętokrzyskie, LDZ – Łódź; KP – Kuyavia-Pomerania; MAS – Masovia; OPO – Opole; LP – Lesser Poland; SUB – Subcarpathia; WP – West Pomerania; LB – Lublin; SL – Silesia; POM – Pomerania; LS – Lower Silesia; LU – Lubusz; WM – Warmia-Masuria

The first group (G I) consisted of the Podlaskie, Świętokrzyskie, Łódź, Greater Poland and Kuyavia-Pomerania voivodeships, where the cattle population is moderately higher (up to 50%) than the national average except for the Greater Poland voivodeship, where it exceeds 300% of the national average (23). In these voivodeships, the seroprevalence of chlamydiosis at animal level was over 3% higher than the national average obtained in this study and it was a predominant factor that determined assignment to this group. Moreover, the herd-level seroprevalence in this group was up to 6% higher than the national average. In this group, congruence between the results for the presence of antibodies against Chlamydia spp. and Coxiella burnetii in the individual cow was a factor of statistical significance.

The second group (G II) consisted of the Opole, Lesser Poland, Subcarpathia and West Pomerania voivodeships, where the dairy cattle population is significantly lower (<50%) than the national average, and the Masovia voivodeship, where dairy cow stock exceeds 300% of the national average. The low seroprevalence level among animals (at least 3% lower than the national average) was a predominant factor that determined assignment to this group. The seroprevalence of Chlamydia spp. at herd level was not uniform and was up to 6% higher than or up to 6% lower than the national average, depending on the voivodeship.

The third group (G III) consisted of the Lublin, Silesia, Pomerania, Lower Silesia and Lubusz voivodeships, which have a moderately lower dairy cattle population (up to 50% below the national average) (23) and these lower stock numbers were a factor that determined assignment to the group. In this group, herd-level seroprevalence was up to 6% lower than the national average, and the seroprevalence among animals ranged from 3% below to 3% above the average.

The fourth group (G IV) consisted of the Warmia-Masuria voivodeship, where none of the analysed factors showed any correlation.

The fifth group (G V) consisted of the Masovia and Greater Poland voivodeships, where the dairy cow population is over 50% higher than the national average (23), and the seroprevalence at herd level exceeded the national average by over 6%. The predominant factor that determined assignment to this group was the size of the population of dairy cows.

A statistically significant correlation was found only between positive results for the presence of Chlamydia spp. and Coxiella burnetii antibodies in the same sera samples in G I. This group consisted of voivodeships where herd seroprevalence was up to 6% higher than the national average obtained in this study, and where there are 50% more head of cattle than the national average.

Analyses conducted for all other factors for G I as well as all factors for G II to G V did not show any statistically significant results.

The presence of Chlamydiaceae DNA in vaginal swabs from seropositive animals was detected only in 2 (1.4%) out of the 142 tested herds. Chlamydiaceae- positive herds were located in Kutnowski county in the Łódź voivodeship. Chlamydiaceae-positive samples were positive in the C. pecorum-specific real-time PCR and yielded threshold values of 31.87 and 35.59. All tested placentas were Chlamydiaceae negative.

The cattle industry in Poland has long been an important branch of the national agricultural sector. In 2022, Poland’s cattle stock was estimated at over 6 million animals, of which over 2 million were dairy cows (20). The highest densities of dairy cattle were noted in Masovia, Great Poland and Podlasie (23). Many infectious agents both viral and bacterial, including intracellular bacteria such as Coxiella burnetii and chlamydiae, might cause economic losses in cattle breeding. Numerous European studies based on serology indicated an endemic distribution of different Chlamydia species in cattle, with prevalence ranging from 5 to 100%, the key investigations being in Ireland 4.75% (29), Germany 19.6% (7), Italy 24% (5), Sweden 28% (9), Austria 45% (19), Switzerland 47% (26) and Germany 100% (2). Data on cattle chlamydiosis in Poland are very limited. This study was conducted to evaluate the epizootic situation of chlamydiosis in dairy cattle herds in Poland via serological testing of sera collected from dairy cows in all voivodeships. A study conducted in the past decade indicated that prevalence of antibodies was quite low, averaging 4.15% in asymptomatic cattle and 7.20% in animals with reproductive disorders (25). Other research including only pregnant and imported cows showed 19.3% seroprevalence (16). The seroprevalence in this study was significantly higher than in previous research, as average TP at herd level being 42.7% (CI 39.8–45.5%) and at animal level 33.3% (CI 21.2 –76.8%). It was nevertheless comparable with the seroprevalence in other European countries, e.g. Switzerland or Austria (19, 26). Differences in seroprevalence between individual voivodeships were revealed in this study (Table 1). Statistical correlations for herd TP were noted, while there was a lack of correlation for TP at animal level (Table 1). According to the OIE Terrestrial Manual, the CFT is suitable only in very limited circumstances for assessing the prevalence of chlamydial infection; therefore, the confidence intervals for TP determined with CFT are very wide, which impacted the statistical analysis. Moreover, chlamydiae share common antigens with other Gram-negative bacteria, making the CFT test or crude ELISAs non-specific (30). Unfortunately, the ELISA test is specific only for C. abortus, and there are no commercially available ELISA kits enabling identification of antibodies to other chlamydial agents. Therefore, only the CFT enables serological screening tests for Chlamydia spp.

A detailed statistical analysis identified five groups of voivodeships, highlighting differences that may have arisen from various factors (Fig. 2). As is commonly known, high stocking density facilitates pathogen transmission, and that was confirmed by the results of the performed analyses. Almost all voivodeships in G I (LDZ, KP) have high (Łódź and Kuyavia-Pomerania) or very high (Greater Poland and Podlaskie) numbers of cattle per 100 ha of agricultural land (23). Those voivodeships as well as Masovia (G II and G V), which also has a very high number of cattle per 100 ha of agricultural land, presented the highest seroprevalence at herd level, up to or over 6% higher than the national average. Moreover, group I was the only group where congruence between positive results for presence of Chlamydia spp. and Coxiella burnetii antibodies in the same sera samples was statistically significant. The lack of a clear correlation between factors in many voivodeships suggests the influence of random factors or other variables that require further investigation. To better assess the impact of potentially significant factors, e.g. herd size, farm management practices, livestock sanitation conditions, intensity of trade and movement of animals, and the impact of other factors e.g. stressors, which may influence disease epidemiology in individual regions, additional studies should be conducted.

A high level of seroprevalence may be the result of cattle exposure to chlamydiae during their lives due to the common circulation of C. pecorum in calves. As cattle grow, they develop a competent immune response, as already reported in sheep, and it might be the reason why chlamydiae shedders were identified only in 2 out of 142 (1.4%) tested herds in this study (3). In contrast to other European studies (18), but similarly to a study from Switzerland (13), we only confirmed the presence of C. pecorum infections and none with other chlamydial species or mixed infections. The single presence of C. pecorum is in accordance with the common hypothesis that the pathogen is endemic in ruminants worldwide (28). Chlamydia pecorum was more commonly detected in Austrian dairy cows than C. abortus, a while German study showed that C. psittaci was the most frequently detected species followed by C. abortus, and C. pecorum was found the most rarely (10, 19). In a previous study from Poland, C. pecorum and C. psittaci were completely absent, whereas C. abortus and C. suis were detected (25). The absence of C. abortus in tested seropositive animals in the present research was surprising, taking into account the type of tested specimens (i.e. vaginal swabs and placentas). The low percentage of Chlamydia spp. shedders is congruent with reports from other European countries, where prevalence of C. pecorum in dairy cows range between 0.7 and 8.9% (10, 19), but notably incongruent with China’s high percentage of 57% (12). Based on Swiss studies, it is known that that C. pecorum loads were higher in younger animals than in older ones (13). In this study only adult dairy cattle were tested, and this might be the explanation for the low number of shedders. However, it is not possible to directly compare this research with other publications because differences abound in many factors, e.g. the animal age and breed, the kind of specimens and the time point of sampling. Comparison of serological results with PCR test results and species identification is impossible because no commercial C. pecorum-specific ELISA tests are available currently. Taking into account the high level of seroprevalence based on CFT results in individual regions in parallel with the rare detection of chlamydial agents, it might be assumed that this pathogen is rarely shed via the tested routes or that the phenomenon of intermittent shedding might occur.

The findings of this research are congruent with previous data on the epizootic situation in cattle worldwide, including Europe and Poland. These data indicate that chlamydial infections are commonly present in Polish dairy cattle across the country but also that there is a much higher level of seroprevalence than in previous years. Chlamydia pecorum was the only detected species, but presence of others cannot be excluded. Further longitudinal studies are necessary on chlamydiae shedding both in dairy and beef cattle herds and also in calves, including different kinds of samples taken from animals in different age categories.