Postpartum dysgalactia syndrome (PDS) in sows is one of the main health concerns characterized by fever, endometritis, and insufficient colostrum and milk production during the first days after farrowing.^1-3 The syndrome decreases growth rate and increases mortality in preweaning piglets due to reduced lactation performance of sows in the first 48 to 72 hours post partum.^1,4,5 It is associated with huge financial losses and negatively affects animal welfare in intensive pig production worldwide.^5,6 The incidence at herd level depends on criteria used to assess the occurrence of PDS and is estimated to vary between 0.5% and 60%, with an average of 13%.^4-8

Although it is considered a multifactorial disease, coliform bacteria such as Escherichia, Enterobacter, Citrobacter, and Klebsiella play a major role in the etiology of the infection.^6,8-11 The importance of Escherichia coli in the clinical manifestation of PDS has been reported in few experimental studies.^10-12 Recent studies demonstrated that endometritis in sows shortly after farrowing is considered to be a risk for development of PDS.^1,3 Additionally, the dominance of E coli in the genital tract of affected sows highlights its potential role in the clinical manifestation of the syndrome.^6,8,13,14 However, to our knowledge, E coli isolates obtained from the vaginal swabs of PDS diseased sows have not been analyzed for presence of virulence genes.

Based on location, clinical diseases, and virulence characteristics, strains of E coli can be classified into intestinal pathogenic E coli (IPEC), extraintestinal pathogenic E coli (ExPEC) and commensal E coli.¹⁵ Enterotoxigenic E coli (ETEC) and shiga toxin-producing E coli (STEC) pathotypes of IPEC in pigs are well demonstrated as etiological agents for diarrhea and edema diseases in piglets.¹⁶ Additionally, urogenital infections caused by uropathogenic E coli (UPEC) and septicemia in pigs are correlated with ExPEC pathotypes.^17,18 In the study of Gerjets et al,¹⁹ E coli isolates from milk samples of healthy sows and sows with coliform mastitis were examined for the presence of virulence genes associated with ExPEC, ETEC, and other pathogenic E coli. Nevertheless, there is a lack of information about the virulence genes of E coli recovered from the genital tract of sows and the occurrence of PDS.

The aim of this study was to determine the presence of virulence genes related to ExPEC and ETEC in vaginal isolates of sows with clinical PDS.

Animal care and use

This work was performed in accordance with the Macedonian Legislation on protection and welfare of animals and approved by the Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Macedonia (Decision No. 0202-418/6).

Materials and methods

Animals

The study was carried out between July 2014 and August 2016 on 5 commercial pig farms in Macedonia. In total, 202 sows with available reproductive data of different parities (1-9) and different genetic lines (Landrace-Yorkshire F1 and Dalland hybrid) were recruited for this study. Sows with unknown reproductive records (parity number and time of farrowing completion) were excluded from the study.

All sows and their litters were clinically examined for the presence of PDS 12 to 24 hours after farrowing (8 am to 10 am) based on predetermined clinical signs (Table 1). During clinical inspection, sows were defined as PDS-affected (PDSA) when they showed pathological vulvar discharge or mastitis and had at least one or more clinical signs listed in Table 1.

After clinical assessment, 47 sows were identified as PDSA, while 155 sows were declared as healthy, or PDS-unaffected (PDSU), and showing none of the clinical signs previously described. On the same day following clinical examination, vaginal swabs were taken for bacteriological testing. In total, 202 samples were taken from 47 PDSA and 155 PDSU sows. Before sampling, the vulva was cleaned and disinfected with 10% iodine solution. Vaginal swabs were taken using sterile metal speculums with 3-cm external diameter and 30 to 40 cm in length, deeply inserted into the vagina, by thorough contact with the ventral mucosa for at least 10 seconds. Swabs were stored at 4°C and transported to the laboratory within 2 hours.

Bacteriological analysis

Bacteriological testing was performed by using routine diagnostic procedures. The initial inoculation of the samples was performed on 5% sheep blood agar (blood agar base; Merck) and selective media for gram-negative bacteria (Xylose Lysine Deoxycholate, MacConkey, and Tryptone Bile X-Glucuronide Agar; Merck). After 24 hours of aerobic incubation at 37°C, the grown bacteria were distinguished by their morphology, hemolysis on blood agar catalase reaction, Gram staining, and growth on selective media. Selected colonies were subcultivated on blood agar for another 24 hours at 37°C to obtain pure cultures. The final identification was performed by automated system VITEK2 Compact (BioMérieux). Obtained E coli isolates were selected for further investigations.

Bacterial DNA of E coli strains was prepared by dissolving 2 to 3 colonies in 200 µL of distilled water. After 30 minutes of heating at 95°C, 2.5 µL of the supernatant was used for polymerase chain reaction (PCR) analyses. In total, 74 E coli isolates were examined using multiplex PCR assays for the presence of 27 virulence genes encoding virulence factors associated with ExPEC, ETEC, and STEC strains as described by Ewers et al¹⁵ and Casey and Bosworth.¹⁶ The complete list of targeted virulence genes and primer sequences used for amplification procedures are shown in Table 2. Avian pathogenic E coli (APEC) strain IMT 2470, UPEC strains¹⁵ IMT7920 and IMT9267, and ETEC strains¹⁶ IMT204, IMT19, IMT4830, and IMT3838 served as controls for molecular assays and were kindly provided by the Institute of Microbiology and Epizootics of the Free University Berlin.

Statistical analysis

Statistical analyses were performed using STATISTICA (version 8.0; StatSoft, Inc). The prevalence of E coli was calculated at sow level and descriptive statistics (Mean [SD]) were applied for the parity of sows positive for E coli. The Mann-Whitney test was used to detect significant differences between the parity of PDSA and PDSU sows positive for E coli. Frequency of detected virulence genes was calculated for E coli isolates in both PDSA and PDSU sows. A Chi square-test and the Fisher’s exact test were preformed to find the differences in the frequency of the E coli virulence genes between PDSA and PDSU sows. The results were considered statistically significant at P < .05.

Multivariable logistic regression was applied to parity and the presence of E coli virulence genes as independent variables with regard to the PDS status. The dependent variable was the occurrence of PDS as a binary trait (PDSA sows or PDSU sows). The presence of the virulence genes and parity with P ≤ .25 were selected to be used in the multivariable logistic model. The final logistic model was developed by following a forward stepwise approach using parity and virulence genes as predictors for PDS. The final model consisted of significant variables with P < .05. The strength of relationships was expressed using odds ratio (OR).

Results

The prevalence of E coli was significantly higher (χ² = 16.287, P < .001) in the PDSA sows (57.45%; 27 of 47) compared to the PDSU sows (26.45%; 41 of 155).The PDSA sows positive for E coli had significantly lower parity (3.18 [1.94]) than the parity observed in PDSU sows (5.21 [2.53]) positive for E coli (U = 293.00; P < .001). From PDSA sows, 33 E coli isolates were recovered from the vaginal swabs, while 41 isolates were detected in vaginal swabs of PDSU sows. Of the 74 E coli isolates, 70 isolates had at least one virulence gene. From the 32 E coli isolates from PDSA sows, 113 virulence genes were identified compared with 138 virulence genes detected in 38 isolates from PDSU sow samples.

The number of virulence genes per isolate ranged from 1 to 10 and 96.41% (242 of 251) of genes were associated with ExPEC strains. The most dominant virulence gene in all E coli isolates was fimC (94.29%; 66 of 70) with prevalence of 90.63% (29 of 32) in isolates of PDSA sows and 97.37% (37 of 38) in isolates from vaginal swabs of PDSU sows. The lowest prevalence of 1.42% was found for K88 (F5) and kpsMTII, while genes irp2, papC, vat, F18, Stx2e, STb, LTI, 987P (F6), K99 (F4), and afa/draB were not found in any of the isolates. Concerning the prevalence of virulence genes between the two groups of sows positive for E coli, fimC was the most dominant gene in both groups of sows, while significance was observed only for iss gene with higher prevalence found in PDSA sow samples (P = .02; Table 3).

Parity and presence of virulence genes iss, iucD, astA, hlyA, sfa/foc, pic, and iha with P ≤ .25 were included in the logistic regression. The multivariable logistic regression model showed that parity associated with the presence of virulence genes iss, sfa/foc, astA, and hlyA were significantly associated with PDS occurrence (R² = 0.373; P < .001; Table 4).

Discussion

In this study, E coli isolates from vaginal tracts of PDSA and PDSU sows were compared to get information on differences in the E coli virulence genes regarding PDS. We found significantly lower parity for PDSA sows positive for E coli in contrast to parity for PDSU sows positive for E coli. This finding is in accordance with the study conducted by Bostedt et al,¹⁴ where E coli was the predominant bacterium in the genital tract of 78 gilts suffering from puerperal septicemia.

Virulence genes belonging to ExPEC strains were the most frequent genes detected in all E coli isolates similar to the results reported by Gerjets et al.¹⁹ Uropathogenic E coli strains, members of ExPEC, are the main causative agents associated with urogenital tract infections (UGTI).^15,20 Colonization of the urogenital tract by UPEC strains are associated with certain virulence genes encoding virulent capsule antigens, iron acquisition systems, adhesions, and secreted toxins.²⁰ The fimC gene, described as a urovirulence factor playing an important role in urinary tract infection,²⁰ was also detected in a high percentage in our study. This finding is in agreement with other studies.^19,21 In a survey conducted by Gerjets et al,¹⁹ fimC was found in 84.7% of the isolates from the milk of sows with coliform mastitis and in 82.3% of the isolates from the milk of healthy sows. Similarly, high prevalence of fimC (91.3%) was found in E coli isolates recovered from sows with UGTI.²¹ In another study, fimC was highly prevalent in E coli isolates obtained from both septicemic and healthy chickens.²² Weak association of the prevalence of fimC and occurrence of PDS was also confirmed in our study where high prevalence (97.4%) of this fimbrial gene was also identified in PDSU sows.

Bacterial serum resistance has been reported as an important virulence factor since it enables bacteria to avoid the bactericidal effect of a serum.²³ Without this virulence factor, bacteria are being lysed by complement, which is more often activated through surface bacterial antigens via an alternative pathway.²⁴ The C3b component plays an important part in the alternative pathway mechanism enabling adherence of bacteria to C5 to C9 bacteriolytic membrane attack complex (MAC).²⁴

Higher prevalence of iss gene in PDSA sows in our research confirms the findings of other researchers.^23-26 Pedersen Mörner et al²³ found that serum resistance virulence factor was more frequently detected in E coli isolates obtained from sows with coliform mastitis. Moreover, this virulence factor was often detected in strains isolated from the milk of mastitic cows.²⁵ Kassé et al²⁶ found that the iss gene was detected in 70% of E coli isolates found in dairy cows with postpartum metritis. The iss gene is more frequently distributed in APEC strains and is closely associated with large transmissible R plasmids or ColV plasmids in APEC (pAPEC-O1, pAPEC-O2-ColBM and pTJ100).²⁷ However, some studies have documented contradictory findings regarding the prevalence of iss between different E coli populations. In the study conducted by Rodriguez-Siek et al,²⁸ iss gene was found in 81% of APEC and in 60% of the UPEC isolates. In addition, this gene was confirmed in 56% of the newborn meningitis E coli (NMEC) strains¹⁵ and in a few human fecal commensal E coli isolates.²⁹ Thus, more frequent presence of iss gene in ExPEC strains may be related to their ability to survive in extraintestinal conditions.²⁹

The virulence genes related to APEC strain IMT2470 that we frequently detected in PDSA sows were found in UPEC strains too.^15,30 In the research of Ewers et al³⁰ at least one virulence gene related with APEC strain IMT2470 was found in all five UPEC isolates. In the survey of Ewers et al,¹⁵ a substantial number of APEC associated genes (iss, iucD, iroN, traT) were also detected in UPEC strains. However, we could not categorize the detected E coli strains into specific ExPEC pathotype by virulence genotyping only. Many virulence features such as iron-uptake systems, protectins, fimbriae, and other adhesins are essential for fitness properties of the bacteria to enable them to efficiently adapt and colonize the host rather than their classical virulence factors primarily included in infection.³¹

In our study, we found that the presence of iss and astA and lower parity increased the clinical manifestation of PDS in sows. Moreover, the adjusted R² of 0.373 obtained for lower parity, presence of iss and astA, and absence of sfa/foc and hlyA were associated with the occurrence of PDS. Scientific data regarding the effect of parity as a risk factor on the occurrence of PDS are inconsistent. While Baer and Bilkei³² reported that higher (> 4) parity sows had increased risk from recidiving mastitis-metritis- agalactia syndrome, other authors found greater risk of postparturient disorders for lower parity sows.^14,33,34

We also found that younger sows vaginally infected by E coli were more prone to disease. According to Hoy,³⁵ higher parity sows have a more developed immune system than lower parity sows primarily due to their wider contact with microbiological agents during their lifetime. Nevertheless, our results could lead to potential biases, such as not having representative samples of virulence factors for the general sow population. This is certainly a weakness of the current study and additional research with adequate sample size is required to determine the biological associations of detected virulence genes and PDS in sows.

In summary, this study found that virulence genes associated with ExPEC were the most frequently detected among E coli isolates recovered from the vaginal swabs of both PDSA and PDSU sows. Lower parity and certain virulence genes related to ExPEC strains were strongly associated with clinical PDS in sows. This study gives novel information about virulence genes of E coli isolated from the genital tract of sows and PDS. The number of sows selected for this research corresponded to the available reproductive data obtained by the commercial pig farms included in the study. However, further research with large and equal sample sizes should be conducted to identify whether specific virulence gene profiles of ExPEC strains recovered from the genital tract are in line with the clinical appearance of PDS. The prevalence of virulence genes from other coliform bacteria and PDS in sows should be considered in future studies.

Implications

Under the conditions of this study:

• Vaginal swabs positive for E coli and iss were associated with PDS in sows.
• Younger sows with certain ExPEC strains were more likely to have clinical PDS.
• Escherichia coli pathotypes could not be categorized by virulence genotyping.

Acknowledgments

Pig farms are acknowledged for their contribution to this study. The authors would like to thank the colleagues from the Laboratories of Molecular Diagnostics and Microbiology from the Faculty of Veterinary Medicine in Skopje for their technical support with the multiplex PCR assays. Dr Antina Lübke-Becker from the Institute of Microbiology and Epizootics of the Free University Berlin is greatly acknowledged for providing control strains for molecular analysis.

Conflict of interest

None reported.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

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