VIRAL IMMUNOLOGY Volume 28, Number 4, 2015 ª Mary Ann Liebert, Inc. Pp. 229–235 DOI: 10.1089/vim.2014.0138
Pathological Evaluation of Reproductive System of Porcine Reproductive and Respiratory Syndrome Virus–Vaccinated and Nonvaccinated Anestrus Sows and Gilts Vasileios Papatsiros,1 Dimitra Psalla,2 Dimitrios Papaioannou,3 Panagiotis Tassis,4 Eleni Tzika,4 and Nikolaos Papaioannou 2
Abstract
The purpose of the study was to evaluate pathologically the reproductive system of nonvaccinated and porcine reproductive and respiratory syndrome virus (PRRSV)-vaccinated sows/gilts, as well as the assessment of vaccination impact on their endometrium/myometrium and ovaries. In a farrow-to-finish farm, all sows and gilts were systematically vaccinated with a PRRSV-inactivated vaccine for a period of 18 months. During the last semester before the beginning of the trial, the genital organs (uterus and ovaries) from 50 females (nonvaccinated primiparous and multiparous sows that did not exhibit signs of estrus until 10 days post-weaning) were collected at the slaughterhouse for gross and microscopic examination (parity 1 to > 6). Respectively, during the following three semesters after the implementation of the experimental PRRSV vaccination scheme, the genital organs from 75 vaccinated sows—25 samples taken along the period of each semester—were also collected. Gross examinations included measurements of uterine horn (thickness and diameter) and ovaries (diameter). Microscopic examination focused on histopathological examination of ovarian cysts or other cystic formations and evaluation of the endometrium and myometrium. Measurements of uterine horn’s thickness/diameter and ovaries’ diameter were at normal levels, and no significant differences were found on gross examination between vaccinated and nonvaccinated females. The results of histopathological examinations did not show lesions of endometritis or myometritis, but revealed ovarian cystic formations with a diameter of > 2 cm that were luteinizing cysts. The presence of these luteinizing cysts was significantly lower in PRRSV-vaccinated animals, compared to nonvaccinated, three semesters ( p = 0.017) after the start of vaccinations and for the total trial period ( p = 0.009).
Introduction
P
orcine Reproductive and Respiratory Syndrome (PRRS) has had a tremendous economic impact on the global pig production since its first appearance in the late 1980s in the United States and Europe (25,37). PRRS virus (PRRSV) infection causes severe reproductive damage on breeding stock and reduces sows’ fertility and longevity (30,39). Reproductive failure is mainly characterized by an increase in premature farrowings and late-term abortions, low farrowing rate, high culling rate, and increased number of abnormal litters (e.g., weak, variably sized, mummified, stillbirths, and dead piglets) (2,6).
The control program of PRRS in breeding stock is based on vaccination with modified live virus (MLV) and killed virus (KV) vaccines (1,27,28,33). Both types of vaccines have beneficial effects on the reproductive performance of vaccinated herds, but generally KV vaccines are considered safer than MLV, as the safety of MLV in sows is questionable (3,10). There are very limited studies, including pathological examinations of PRRSV naturally or experimentally infected sows, in the recent literature. Moreover, there are no detailed published data about pathological examinations in PRRSV-vaccinated sows or gilts. Microscopic lesions are frequently observed in the uterus of naturally or experimentally PRRSV-infected pregnant sows and myometritis,
1
Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece. Department of Pathology, 4Farm Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece. 3 Directorate General of Veterinary Services, Ministry of Rural Development and Food, Athens, Greece. 2
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endometritis, placentitis, and microseparations in the maternal fetal unit have been previously described (5,20,34). Microscopic lesions of pleocellular endometritis and myometritis and myometrial and endometrial edema have been reported in experimental PRRSV-infected sows (20). Moreover, lymphohistiocytic vasculitis and perivascular cell infiltration have been referred in the endometrium of both naturally and experimentally infected sows (34). However, the authors’ previous studies have indicated that vaccination of breeding stock with inactivated vaccine leads to an improvement in the reproductive parameters at farm level, as well as to significant a reduction in the culling rate due to reproductive failure (27,28). The aim of the present field study was to evaluate pathologically the reproductive system of PRRSV-vaccinated and nonvaccinated females, assessing the vaccination impact on their endometrium/myometrium and ovaries. Materials and Methods
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follows: (a) basic pregnancy feed: digestible energy 13.1 Mj/kg, crude protein 14.0%, fiber 5.5%, lysine 0.9%, calcium 0.96%, and total phosphorus 0.73%; (b) basic lactation feed: digestible energy 14.2 Mj/kg, crude protein 17.0%, fiber 5.4%, lysine 1.0%, calcium 0.95%, and total phosphorus 0.82%. History of the trial farm
The farm suffered an acute PRRSV infection 5 years prior to the initiation of the trial. Since then, chronic course of the disease with several breakthroughs during each year had been observed. Diagnosis was based on clinical signs and subsequent serology. A year prior to the initiation of the vaccination trial, the farm still had low reproductive performance as evidenced by increased return-to-estrus rates, small litters, weak piglets, and increased piglet mortality (28). After the diagnosis of PRRS 5 years ago, no vaccination against PRRS was applied to the farm.
Experimental material
Experimental design
The commercial inactivated Progressis vaccine (Merial SAS, France), based on the European P120 strain, was used. The vaccine dose contained ‡ 102.5 IF units and was suspended in 2 mL of an oily adjuvant (containing hydrogenated polyisorbutene) for intramuscular injection behind the ear.
Initially, all sows were simultaneously vaccinated (first vaccination), except those being 1 week prior to 2 weeks after service (these females were primarily vaccinated 3 weeks later). All previously vaccinated animals received a booster vaccination between 55 and 60 days of next gestation, and thereafter at each gestation for a period of 18 months (three semesters). The replacement gilts were vaccinated twice prior to breeding (first vaccination, not earlier than 170 days old) and boostered in each pregnancy, as described previously. All procedures during this clinical study were carried out according to the Code of Practice for the Conduct of Clinical trials for Veterinary Medical Products, and the animals were maintained in accordance with national and European animal welfare requirements (13,26). In addition, the study was performed under license for experimenting on animals from the local Veterinary Administration Office (License No. 07/1855). Applied management and vaccination programs, as well as the nutrient specification and feeding schedule, remained the same from at least 1 year before until the end of the study.
Trial farm
The trial was performed in a commercial farrow-to-finish site with a capacity of 900 sows under production, located in northern Greece. A grandparent nucleus of 70 sows was kept in the farm for producing its own gilts. The farm facilities included four farrowing houses, five flat-deck units, six growing houses, six finishing houses, four mating-pregnancy (dry period) stables, one breeding stock house, a feed mill, and an artificial insemination (AI) laboratory. Animals were accommodated in appropriate housing and microclimate conditions, while each site of the production facilities was filled and emptied in an all-in/all-out manner, with thorough cleaning and disinfection between subsequent batches. The trial farm applied an AI program with raw semen, and females were inseminated twice with fresh semen from the same boar. Semen collection, dilution, and storage were performed at the farm All sows/gilts of the farm were vaccinated against Aujeszky’s disease (AD), swine influenza (SI), parvovirus infection, atrophic rhinitis (AR), erysipelas, Escherichia coli, and Clostridium perfringens infections (type A and C). All boars were vaccinated every 6 months against erysipelas, AD, and SI. Fatteners were vaccinated against AD and SI, and weaners against Mycoplasma hyopneumoniae. For the antiparasitic control, all breeding females were treated with a single ivermectin injection 14 days prior to each farrowing, while the boars were treated twice a year. Routine serological Leptospira spp. testing in blood samples of pigs from all ages, as well as mycotoxicological analysis (zearalenone, aflatoxines, deoxynivalenol, nivalenol) in feed raw materials, was performed. The feed provided to the animals was self-prepared based on a corn/barley/wheat–soya meal, depending on the season. Prior to and during the study, gilts/sows were fed with two types of feed, the nutrient specification of which were as
Sampling schedule—examinations
During the last semester before the beginning of the trial, the genital organs (uterus and ovaries) from 50 females (apparently healthy, nonvaccinated primiparous and multiparous sows that did not exhibit signs of estrus until 10 days post weaning) were collected at the slaughterhouse for gross and microscopic examinations. Respectively, during the following three semesters after the implementation of the experimental PRRSV vaccination scheme, the genital organs from 75 vaccinated females (as previously described)— 25 samples taken along the period of each semester—were also collected. Special care was taken in order to ensure sufficient homogeneity of parity distribution among each semester sampling procedure, prior and especially during the trial (Table 1). Within the framework of the macroscopic evaluation of the genital organs, the uterus (body and horns) and the ovaries of each sampled female were weighed, ovarian
PATHOLOGICAL EVALUATION OF PRRSV-VACCINATED SOWS
Table 1. Parity Distribution of Sampled Females During the Study Period Sampling semester relative to the start of vaccinations
Table 2. Prevalence of PRRSV-Seropositive Nonvaccinated Females Over the Study Period
Parity distribution 1 2
3
4
5
-1 Nonvaccinated 6 8 12 10 10 Vaccinated +1 4 4 5 6 5 +2 2 4 6 4 6 +3 3 5 6 5 5 + 1 to + 3 9 13 17 15 16
Number of seropositive* animals/total number of animals sampled (%)
> 6 Total 4 1 3 1 5
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50* 25 25 25 75**
*50 = total nonvaccinated sampled females before the beginning of the trial. **75 = total vaccinated sampled females 18 months after the implementation of the PRRSV vaccination scheme. PRRSV, porcine reproductive and respiratory syndrome virus.
formations were characterized and counted, the measurements of the uterine horns (thickness and diameter) and ovaries (diameter) were performed, and the presence of any existing gross lesion (other than ovarian cystic formation) was recorded. Microscopic examination focused on histopathological examination of ovarian cystic formations, as well as of the endometrium and myometrium. For the measurements of the genital organs, a vernier caliper was used, immediately after the cutting of carcasses. In each uterine horn, 10 different measurements at 10 different points were recorded, and the average thickness and the maximum diameter of each horn were calculated. The vernier caliper used provided a precision of 0.01 mm (10 lm). In addition, the maximum diameter of each ovary (left and right) was measured, as was the diameter of follicles/cysts or other visible clusters that were recorded in the ovaries. For the histopathological examination, tissue samples were fixed in buffered 10% formalin solution for 48 h, embedded in paraffin, cut (sections 4–6 l thick), and stained with hematoxylin and eosin. Time from tissue collection to tissue placement in the fixative ranged from 10 to 20 min. Moreover, blood samples from 15 nonvaccinated sows (3 per parity 1–5) and 9 replacement gilts were collected during the last month before the beginning of the trial. Subsequently, 56 blood samples from replacement gilts (23–24 weeks old) were collected at 6, 12, and 18 months after the implementation of the experimental PRRSV vaccination scheme. All serum samples were examined for PRRSV-specific antibody titers using the immunoperoxidase monolayer assay (IPMA) technique, described by Wensvoort et al. (36). The lower positive dilution of the test was 1:40. Data analysis
Each animal per sampling group was considered as the experimental unit. Data were subjected to one-way analysis of variance using the general linear model of the SAS system (v8.01; SAS Institute, Inc., Cary, NC). The normality of the data was tested with the Kolmogorov–Smirnov test, and the homogeneity of variances with Levene’s test. The t-test was used to compare means, and Pearson’s chi-square analysis was performed to compare the parameters expressed as frequencies. In cases where the trans-
Sampling time relative to the start of vaccinations
Sows (parity 1 to 5)
1 month prior 6 months post 12 months post 18 months post
11/15 (73.3)
Gilts 9/9 (100) 19/19 (100) 14/15 (93.3) 21/22 (95.5)
*Females with PRRSV antibody titers > 40 (as detected by immunoperoxidase monolayer assay).
formations of real values did not bring about the expected homogeneity of variations, the Kruskall–Wallis test was used. In all cases, the significance was attained if the p-value was < 0.05. Results Routine Leptospira spp. and mycotoxin examinations
Laboratory examinations during the study (one semester prior to three semesters after the initiation of the trial) did not reveal Leptospira spp. antibody titers or the presence of mycotoxins in feed raw materials at detectable levels (data not shown). Serological results
The results related to the evaluation of the serological profile (prevalence of PRRSV-seropositive females) of the farm over the study period are presented in Table 2. One month prior to the start of vaccination, 73.3% of the sows were found to be infected with PRRSV, whilst the respective percentage of the sampled gilts was 100%. During the next 18 months after the implementation of the PRRSV vaccination scheme, most unvaccinated sampled gilts were found to have specific antibodies in their blood, and the prevalence of PRRSV-seropositive gilts remained constantly > 93.3%. Gross and histopathological examinations
Gross examinations did not reveal significant differences between PRRSV-vaccinated and nonvaccinated females, based on the measurements of uterine horn’s thickness/diameter and ovaries’ diameter, which were in the normal range (Table 3). However, during gross examination, the presence of cystic formations with a diameter > 2 cm was observed. The histopathological examinations indicated that these cystic formations of uterus (with diameter > 2 cm) were luteinizing cysts (Figs. 1 and 2). The presence of luteinizing cysts was significantly lower in PRRSV-vaccinated animals ( p = 0.017) three semesters after the start of vaccinations and for the total trial period of 18 months ( p = 0.009) compared with nonvaccinated animals (for the period of one semester prior the start of vaccinations; Table 4). The histopathological examinations did not revealed lesions of endometritis or myometritis.
2.53 – 0.69 2.52 – 0.53 2.56 – 0.69 2.55 – 0.58 3.78 – 0.44 3.93 – 0.51 4.19 – 0.45 4.12 – 0.47
2.52 – 0.68 2.51 – 0.67 2.54 – 0.63 2.56 – 0.56
FIG. 1. a sow.
3.95 – 0.45 4.46 – 0.66 3.89 – 0.50 4.39 – 0.45
Left Right
Multiple luteinized follicular cysts in the ovary of
0.78 – 0.22 0.77 – 0.28 0.77 – 0.24 0.76 – 0.25
0.76 – 0.27 0.74 – 0.30 0.76 – 0.28 0.77 – 0.26
Discussion
Means within the same column do not differ significantly ( p > 0.05).
2.54 – 0.73 2.56 – 0.87 2.53 – 0.75 2.56 – 0.61 2.53 – 0.77 2.55 – 0.85 2.56 – 0.69 2.55 – 0.66 806.2 – 185.4 788.5 – 222.1 832.7 – 201.6 814.8 – 189.3 Unvaccinated Vaccinated (n = 50) (n = 25) (n = 25) (n = 25) -1 +1 +2 +3
Left Right Left
Weight of uterine body and horns (g) (mean – SD)
Right Left
Weight of ovaries (g) (mean – SD) Maximum diameter of uterine horns (cm) (mean – SD) Thickness of uterine horns (mm) (mean – SD)
Sampling semester relative to the start of vaccinations (number of sampled females)
Gross examination of genital organs
Table 3. Gross Examination of Genital Organs from PRRSV-Vaccinated and Nonvaccinated Females
Right
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Diameter of ovaries (cm) (mean – SD)
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The occurrence of ovarian cysts affects overall reproductive performance, resulting in prolonged anestrus and thus in complete infertility (15). Moreover, ovarian cysts are a common cause of noninfectious infertility that leads to abnormal estrus behavior (4). Their size and number can vary significantly (11,32). The incidence of cystic ovaries in culled sows and gilts, due to infertility problems, varies from 1.7% to 24.0% (12,16,32,36). Generally, in PRRSV-infected farms, sows or gilts farrowing abnormal litters and periparturient mortality are usually noticed (9,18). Typically, when these females are bred again, they can exhibit delayed returns to estrus and suffer low conception rates. These low conception rates, combined with the previously mentioned abnormalities, increase abortion and irregular returns to estrus rates, as well as the number of nonpregnant sows, resulting in a depression in farrowing rate for a complete reproductive cycle. Similarly to previous studies, no significant differences were noticed in the present study between PRRSV-vaccinated and nonvaccinated animals concerning gross measurements of ovaries and uterus (21).
FIG. 2. Wall of a luteinizing cyst consisted by partially luteinized cells and a thick layer of fibrous tissue. Hematoxylin and eosin staining. Magnification 200 · .
PATHOLOGICAL EVALUATION OF PRRSV-VACCINATED SOWS
233
Table 4. Prevalence of PRRSV-Vaccinated and Nonvaccinated Sows with Inactive or Cystic Ovaries Sampling semester relative to the start of vaccinations Number of cases/total number of sows sampled (%) Sows with inactive ovaries* Sows with cystic ovaries**
Prior -1 8/50 (16.0) 8/50 (16.0) 14/50 (28.0)*** 14/50 (28.0)a
Post +1 2/25 (8.0) 4/25 (16.0)***
+2
+3
5/25 (20.0) 10/75 (13.3) 5/25 (20.0)*** 11/75 (14.7)a
3/25 (12.0) 2/25 (8.0)***
*Defined as sows presenting no corpora lutea and size of follicles £ 4 mm or none, in both ovaries. **Defined as sows presenting at least one cystic formation ‡ 15 mm and no corpora lutea, in any of their ovaries. ***Significantly different percentages ( p < 0.05). a p = 0.068.
The myometrium and/or endometrium of PRRSVinfected sows are edematous with lymphohistiocytic perivascular cuffs, and less commonly there are segmental lymphohistiocytic vasculitis in small vessels and microseperations between endometrial epithelium and trophoblasts (37). The histopathological results did not reveal lesions in the endometrium or myometrium in our study. On the contrary, in a previous study, multiple foci of lymphoplasmacytic inflammation were observed in the myometrium of sows after experimental infection with PRRSV (5). Furthermore, Lager and Halbur observed pleocellular endometritis and myometritis in uncomplicated PRRSV-induced abortion cases, suggesting that these uterine findings were also PRRSV induced (20). Moreover, myometritis, endometritis, and placentitis of the maternal placenta have been observed in experimentally and naturally infected sows (34). The design of this study was based on previous clinical observations at the slaughterhouse, which showed that the number of culled females from breeding stock due to ovarian cysts were higher in endemically PRRSV-infected farms when compared to other farms (31). The analysis of data of those farms showed that the main cause of early culling was fertility problems after weaning. In live animals, follicles maintained with a diameter of ‡ 2.0 cm for > 5 days after the onset of estrus have been regarded as cystic ovaries (4). Multiple large or small cysts without corpora lutea in the ovaries are common and are always associated with temporary or permanent infertility (16). Generally, in clinical practice, most sows with cystic ovaries are culled due to their failure to farrow or after repeat mating without conception. This problem leads to an increase in the nonproductive days of the breeding herd. Sows with ovarian cysts have been reported to have a higher incidence rate of return-to-estrus (34% vs. 7.7%) and anestrus sows that were not pregnant (10.6% vs. 0.6%), as well as a lower farrowing rate (52.2% vs. 90.0%) than normal sows (4). Most of the sows with multiple large cysts show intermittent or permanent anestrus. Previous studies have reported a link between reproductive problems and the presence of ovarian cysts found at the slaughterhouse (14,16,23,24). Additionally, ovarian cysts are a cause of lower conception rates, irregular estrous cycles, or behavioral changes, and they are present in approximately 10% of culled sows due to fertility problems (16,24). Generally, ovarian cysts in sows do not induce pathognomic clinical signs, other than an irregular and/or prolonged estrus cycle, permanent anestrus, and lower fer-
tility (4,17,32). In clinical practice, the use of gonadotropin PG600 (400 IU of equine chorionic gonadotropin and 200 IU of human chorionic gonadotropin) to anestrus sows that have not displayed estrus by day 7 post-weaning in PRRSV-infected farms could be a useful tool to increase estrus response in weaned sows (22). In addition, it is seems there is an essential relation between the presence of ovarian cysts and histological changes in the ovaries and oviductal and uterine mucosa, which eventually causes reproductive organ dysfunctions (36). If this state lasts for long, it may lead to permanent changes in the genital tract and result in persistent infertility of the sows, as well as an increase in the culling rate. The occurrence of luteinizing cysts in the present study was significantly lower in PRRSV-vaccinated animals ( p = 0.017) three semesters after the start of vaccinations and for the total trial period of 18 months ( p = 0.009). The results of the present study confirm the clinical observations of Papatsiros that ovarian cysts were more often observed in culled females from PRRSV endemic areas, as the occurrence of these luteinizing cysts were significantly decreased in PRRSV-vaccinated sows and gilts (31). The low female culling rate is an important management factor for the health status, economy, and production level of swine farms (19). In the case of PRRSV-infected farms, an increase of female culling rate occurs due to reproductive problems (30,39). A high removal rate requires a higher replacement rate, and this increases the number of nonproductive sow days, resulting in a higher pig production cost (7,8). Moreover, in a previous study of our group, the use of the same inactivated vaccine in sows and gilts proved to reduce the negative effects of PRRSV infection, improving significantly their reproductive performance and their litter characteristics (9). In addition, in another study, it is indicated that the same vaccination scheme of breeding stock leads to a significant reduction of culling rate due to reproductive failure 1.5 years after the start of vaccination (29). In conclusion, ovarian cysts are included in the causes of impaired fertility in endemic or chronic cases of PRRS. Based on the results of this study, PRRSV vaccination of breeding stock with an inactivated vaccine positively affects their reproductive system, as it is linked with a reduction in the occurrence of cystic formations (luteinizing cysts) in the ovaries (diameter > 2 cm). The results of this study are based on samples from a large-scale endemically PRRSVinfected farm. However, it would be interesting for future
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field trials to collect samples from PRRSV-vaccinated and nonvaccinated sows and replacement gilts from farms in different areas after initial infection or outbreak with PRRSV. The outcome of such follow-up studies would add valuable information on porcine health management that swine practitioners should apply in PRRSV-infected farms. Acknowledgments
This work was supported by Merial SAS through the Research Committee of the Aristotle University of Thessaloniki (Code no: 20567, Scientific Responsible S.C. Kyriakis). Author Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Dr. Vasileios G. Papatsiros Clinic of Medicine Faculty of Veterinary Medicine University of Thessaly 43100 Karditsa Greece E-mail: [email protected]
Pathological Evaluation of Reproductive System of Porcine Reproductive and Respiratory Syndrome Virus–Vaccinated and Nonvaccinated Anestrus Sows and Gilts Vasileios Papatsiros,1 Dimitra Psalla,2 Dimitrios Papaioannou,3 Panagiotis Tassis,4 Eleni Tzika,4 and Nikolaos Papaioannou 2
Abstract
The purpose of the study was to evaluate pathologically the reproductive system of nonvaccinated and porcine reproductive and respiratory syndrome virus (PRRSV)-vaccinated sows/gilts, as well as the assessment of vaccination impact on their endometrium/myometrium and ovaries. In a farrow-to-finish farm, all sows and gilts were systematically vaccinated with a PRRSV-inactivated vaccine for a period of 18 months. During the last semester before the beginning of the trial, the genital organs (uterus and ovaries) from 50 females (nonvaccinated primiparous and multiparous sows that did not exhibit signs of estrus until 10 days post-weaning) were collected at the slaughterhouse for gross and microscopic examination (parity 1 to > 6). Respectively, during the following three semesters after the implementation of the experimental PRRSV vaccination scheme, the genital organs from 75 vaccinated sows—25 samples taken along the period of each semester—were also collected. Gross examinations included measurements of uterine horn (thickness and diameter) and ovaries (diameter). Microscopic examination focused on histopathological examination of ovarian cysts or other cystic formations and evaluation of the endometrium and myometrium. Measurements of uterine horn’s thickness/diameter and ovaries’ diameter were at normal levels, and no significant differences were found on gross examination between vaccinated and nonvaccinated females. The results of histopathological examinations did not show lesions of endometritis or myometritis, but revealed ovarian cystic formations with a diameter of > 2 cm that were luteinizing cysts. The presence of these luteinizing cysts was significantly lower in PRRSV-vaccinated animals, compared to nonvaccinated, three semesters ( p = 0.017) after the start of vaccinations and for the total trial period ( p = 0.009).
Introduction
P
orcine Reproductive and Respiratory Syndrome (PRRS) has had a tremendous economic impact on the global pig production since its first appearance in the late 1980s in the United States and Europe (25,37). PRRS virus (PRRSV) infection causes severe reproductive damage on breeding stock and reduces sows’ fertility and longevity (30,39). Reproductive failure is mainly characterized by an increase in premature farrowings and late-term abortions, low farrowing rate, high culling rate, and increased number of abnormal litters (e.g., weak, variably sized, mummified, stillbirths, and dead piglets) (2,6).
The control program of PRRS in breeding stock is based on vaccination with modified live virus (MLV) and killed virus (KV) vaccines (1,27,28,33). Both types of vaccines have beneficial effects on the reproductive performance of vaccinated herds, but generally KV vaccines are considered safer than MLV, as the safety of MLV in sows is questionable (3,10). There are very limited studies, including pathological examinations of PRRSV naturally or experimentally infected sows, in the recent literature. Moreover, there are no detailed published data about pathological examinations in PRRSV-vaccinated sows or gilts. Microscopic lesions are frequently observed in the uterus of naturally or experimentally PRRSV-infected pregnant sows and myometritis,
1
Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece. Department of Pathology, 4Farm Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece. 3 Directorate General of Veterinary Services, Ministry of Rural Development and Food, Athens, Greece. 2
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endometritis, placentitis, and microseparations in the maternal fetal unit have been previously described (5,20,34). Microscopic lesions of pleocellular endometritis and myometritis and myometrial and endometrial edema have been reported in experimental PRRSV-infected sows (20). Moreover, lymphohistiocytic vasculitis and perivascular cell infiltration have been referred in the endometrium of both naturally and experimentally infected sows (34). However, the authors’ previous studies have indicated that vaccination of breeding stock with inactivated vaccine leads to an improvement in the reproductive parameters at farm level, as well as to significant a reduction in the culling rate due to reproductive failure (27,28). The aim of the present field study was to evaluate pathologically the reproductive system of PRRSV-vaccinated and nonvaccinated females, assessing the vaccination impact on their endometrium/myometrium and ovaries. Materials and Methods
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follows: (a) basic pregnancy feed: digestible energy 13.1 Mj/kg, crude protein 14.0%, fiber 5.5%, lysine 0.9%, calcium 0.96%, and total phosphorus 0.73%; (b) basic lactation feed: digestible energy 14.2 Mj/kg, crude protein 17.0%, fiber 5.4%, lysine 1.0%, calcium 0.95%, and total phosphorus 0.82%. History of the trial farm
The farm suffered an acute PRRSV infection 5 years prior to the initiation of the trial. Since then, chronic course of the disease with several breakthroughs during each year had been observed. Diagnosis was based on clinical signs and subsequent serology. A year prior to the initiation of the vaccination trial, the farm still had low reproductive performance as evidenced by increased return-to-estrus rates, small litters, weak piglets, and increased piglet mortality (28). After the diagnosis of PRRS 5 years ago, no vaccination against PRRS was applied to the farm.
Experimental material
Experimental design
The commercial inactivated Progressis vaccine (Merial SAS, France), based on the European P120 strain, was used. The vaccine dose contained ‡ 102.5 IF units and was suspended in 2 mL of an oily adjuvant (containing hydrogenated polyisorbutene) for intramuscular injection behind the ear.
Initially, all sows were simultaneously vaccinated (first vaccination), except those being 1 week prior to 2 weeks after service (these females were primarily vaccinated 3 weeks later). All previously vaccinated animals received a booster vaccination between 55 and 60 days of next gestation, and thereafter at each gestation for a period of 18 months (three semesters). The replacement gilts were vaccinated twice prior to breeding (first vaccination, not earlier than 170 days old) and boostered in each pregnancy, as described previously. All procedures during this clinical study were carried out according to the Code of Practice for the Conduct of Clinical trials for Veterinary Medical Products, and the animals were maintained in accordance with national and European animal welfare requirements (13,26). In addition, the study was performed under license for experimenting on animals from the local Veterinary Administration Office (License No. 07/1855). Applied management and vaccination programs, as well as the nutrient specification and feeding schedule, remained the same from at least 1 year before until the end of the study.
Trial farm
The trial was performed in a commercial farrow-to-finish site with a capacity of 900 sows under production, located in northern Greece. A grandparent nucleus of 70 sows was kept in the farm for producing its own gilts. The farm facilities included four farrowing houses, five flat-deck units, six growing houses, six finishing houses, four mating-pregnancy (dry period) stables, one breeding stock house, a feed mill, and an artificial insemination (AI) laboratory. Animals were accommodated in appropriate housing and microclimate conditions, while each site of the production facilities was filled and emptied in an all-in/all-out manner, with thorough cleaning and disinfection between subsequent batches. The trial farm applied an AI program with raw semen, and females were inseminated twice with fresh semen from the same boar. Semen collection, dilution, and storage were performed at the farm All sows/gilts of the farm were vaccinated against Aujeszky’s disease (AD), swine influenza (SI), parvovirus infection, atrophic rhinitis (AR), erysipelas, Escherichia coli, and Clostridium perfringens infections (type A and C). All boars were vaccinated every 6 months against erysipelas, AD, and SI. Fatteners were vaccinated against AD and SI, and weaners against Mycoplasma hyopneumoniae. For the antiparasitic control, all breeding females were treated with a single ivermectin injection 14 days prior to each farrowing, while the boars were treated twice a year. Routine serological Leptospira spp. testing in blood samples of pigs from all ages, as well as mycotoxicological analysis (zearalenone, aflatoxines, deoxynivalenol, nivalenol) in feed raw materials, was performed. The feed provided to the animals was self-prepared based on a corn/barley/wheat–soya meal, depending on the season. Prior to and during the study, gilts/sows were fed with two types of feed, the nutrient specification of which were as
Sampling schedule—examinations
During the last semester before the beginning of the trial, the genital organs (uterus and ovaries) from 50 females (apparently healthy, nonvaccinated primiparous and multiparous sows that did not exhibit signs of estrus until 10 days post weaning) were collected at the slaughterhouse for gross and microscopic examinations. Respectively, during the following three semesters after the implementation of the experimental PRRSV vaccination scheme, the genital organs from 75 vaccinated females (as previously described)— 25 samples taken along the period of each semester—were also collected. Special care was taken in order to ensure sufficient homogeneity of parity distribution among each semester sampling procedure, prior and especially during the trial (Table 1). Within the framework of the macroscopic evaluation of the genital organs, the uterus (body and horns) and the ovaries of each sampled female were weighed, ovarian
PATHOLOGICAL EVALUATION OF PRRSV-VACCINATED SOWS
Table 1. Parity Distribution of Sampled Females During the Study Period Sampling semester relative to the start of vaccinations
Table 2. Prevalence of PRRSV-Seropositive Nonvaccinated Females Over the Study Period
Parity distribution 1 2
3
4
5
-1 Nonvaccinated 6 8 12 10 10 Vaccinated +1 4 4 5 6 5 +2 2 4 6 4 6 +3 3 5 6 5 5 + 1 to + 3 9 13 17 15 16
Number of seropositive* animals/total number of animals sampled (%)
> 6 Total 4 1 3 1 5
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50* 25 25 25 75**
*50 = total nonvaccinated sampled females before the beginning of the trial. **75 = total vaccinated sampled females 18 months after the implementation of the PRRSV vaccination scheme. PRRSV, porcine reproductive and respiratory syndrome virus.
formations were characterized and counted, the measurements of the uterine horns (thickness and diameter) and ovaries (diameter) were performed, and the presence of any existing gross lesion (other than ovarian cystic formation) was recorded. Microscopic examination focused on histopathological examination of ovarian cystic formations, as well as of the endometrium and myometrium. For the measurements of the genital organs, a vernier caliper was used, immediately after the cutting of carcasses. In each uterine horn, 10 different measurements at 10 different points were recorded, and the average thickness and the maximum diameter of each horn were calculated. The vernier caliper used provided a precision of 0.01 mm (10 lm). In addition, the maximum diameter of each ovary (left and right) was measured, as was the diameter of follicles/cysts or other visible clusters that were recorded in the ovaries. For the histopathological examination, tissue samples were fixed in buffered 10% formalin solution for 48 h, embedded in paraffin, cut (sections 4–6 l thick), and stained with hematoxylin and eosin. Time from tissue collection to tissue placement in the fixative ranged from 10 to 20 min. Moreover, blood samples from 15 nonvaccinated sows (3 per parity 1–5) and 9 replacement gilts were collected during the last month before the beginning of the trial. Subsequently, 56 blood samples from replacement gilts (23–24 weeks old) were collected at 6, 12, and 18 months after the implementation of the experimental PRRSV vaccination scheme. All serum samples were examined for PRRSV-specific antibody titers using the immunoperoxidase monolayer assay (IPMA) technique, described by Wensvoort et al. (36). The lower positive dilution of the test was 1:40. Data analysis
Each animal per sampling group was considered as the experimental unit. Data were subjected to one-way analysis of variance using the general linear model of the SAS system (v8.01; SAS Institute, Inc., Cary, NC). The normality of the data was tested with the Kolmogorov–Smirnov test, and the homogeneity of variances with Levene’s test. The t-test was used to compare means, and Pearson’s chi-square analysis was performed to compare the parameters expressed as frequencies. In cases where the trans-
Sampling time relative to the start of vaccinations
Sows (parity 1 to 5)
1 month prior 6 months post 12 months post 18 months post
11/15 (73.3)
Gilts 9/9 (100) 19/19 (100) 14/15 (93.3) 21/22 (95.5)
*Females with PRRSV antibody titers > 40 (as detected by immunoperoxidase monolayer assay).
formations of real values did not bring about the expected homogeneity of variations, the Kruskall–Wallis test was used. In all cases, the significance was attained if the p-value was < 0.05. Results Routine Leptospira spp. and mycotoxin examinations
Laboratory examinations during the study (one semester prior to three semesters after the initiation of the trial) did not reveal Leptospira spp. antibody titers or the presence of mycotoxins in feed raw materials at detectable levels (data not shown). Serological results
The results related to the evaluation of the serological profile (prevalence of PRRSV-seropositive females) of the farm over the study period are presented in Table 2. One month prior to the start of vaccination, 73.3% of the sows were found to be infected with PRRSV, whilst the respective percentage of the sampled gilts was 100%. During the next 18 months after the implementation of the PRRSV vaccination scheme, most unvaccinated sampled gilts were found to have specific antibodies in their blood, and the prevalence of PRRSV-seropositive gilts remained constantly > 93.3%. Gross and histopathological examinations
Gross examinations did not reveal significant differences between PRRSV-vaccinated and nonvaccinated females, based on the measurements of uterine horn’s thickness/diameter and ovaries’ diameter, which were in the normal range (Table 3). However, during gross examination, the presence of cystic formations with a diameter > 2 cm was observed. The histopathological examinations indicated that these cystic formations of uterus (with diameter > 2 cm) were luteinizing cysts (Figs. 1 and 2). The presence of luteinizing cysts was significantly lower in PRRSV-vaccinated animals ( p = 0.017) three semesters after the start of vaccinations and for the total trial period of 18 months ( p = 0.009) compared with nonvaccinated animals (for the period of one semester prior the start of vaccinations; Table 4). The histopathological examinations did not revealed lesions of endometritis or myometritis.
2.53 – 0.69 2.52 – 0.53 2.56 – 0.69 2.55 – 0.58 3.78 – 0.44 3.93 – 0.51 4.19 – 0.45 4.12 – 0.47
2.52 – 0.68 2.51 – 0.67 2.54 – 0.63 2.56 – 0.56
FIG. 1. a sow.
3.95 – 0.45 4.46 – 0.66 3.89 – 0.50 4.39 – 0.45
Left Right
Multiple luteinized follicular cysts in the ovary of
0.78 – 0.22 0.77 – 0.28 0.77 – 0.24 0.76 – 0.25
0.76 – 0.27 0.74 – 0.30 0.76 – 0.28 0.77 – 0.26
Discussion
Means within the same column do not differ significantly ( p > 0.05).
2.54 – 0.73 2.56 – 0.87 2.53 – 0.75 2.56 – 0.61 2.53 – 0.77 2.55 – 0.85 2.56 – 0.69 2.55 – 0.66 806.2 – 185.4 788.5 – 222.1 832.7 – 201.6 814.8 – 189.3 Unvaccinated Vaccinated (n = 50) (n = 25) (n = 25) (n = 25) -1 +1 +2 +3
Left Right Left
Weight of uterine body and horns (g) (mean – SD)
Right Left
Weight of ovaries (g) (mean – SD) Maximum diameter of uterine horns (cm) (mean – SD) Thickness of uterine horns (mm) (mean – SD)
Sampling semester relative to the start of vaccinations (number of sampled females)
Gross examination of genital organs
Table 3. Gross Examination of Genital Organs from PRRSV-Vaccinated and Nonvaccinated Females
Right
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Diameter of ovaries (cm) (mean – SD)
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The occurrence of ovarian cysts affects overall reproductive performance, resulting in prolonged anestrus and thus in complete infertility (15). Moreover, ovarian cysts are a common cause of noninfectious infertility that leads to abnormal estrus behavior (4). Their size and number can vary significantly (11,32). The incidence of cystic ovaries in culled sows and gilts, due to infertility problems, varies from 1.7% to 24.0% (12,16,32,36). Generally, in PRRSV-infected farms, sows or gilts farrowing abnormal litters and periparturient mortality are usually noticed (9,18). Typically, when these females are bred again, they can exhibit delayed returns to estrus and suffer low conception rates. These low conception rates, combined with the previously mentioned abnormalities, increase abortion and irregular returns to estrus rates, as well as the number of nonpregnant sows, resulting in a depression in farrowing rate for a complete reproductive cycle. Similarly to previous studies, no significant differences were noticed in the present study between PRRSV-vaccinated and nonvaccinated animals concerning gross measurements of ovaries and uterus (21).
FIG. 2. Wall of a luteinizing cyst consisted by partially luteinized cells and a thick layer of fibrous tissue. Hematoxylin and eosin staining. Magnification 200 · .
PATHOLOGICAL EVALUATION OF PRRSV-VACCINATED SOWS
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Table 4. Prevalence of PRRSV-Vaccinated and Nonvaccinated Sows with Inactive or Cystic Ovaries Sampling semester relative to the start of vaccinations Number of cases/total number of sows sampled (%) Sows with inactive ovaries* Sows with cystic ovaries**
Prior -1 8/50 (16.0) 8/50 (16.0) 14/50 (28.0)*** 14/50 (28.0)a
Post +1 2/25 (8.0) 4/25 (16.0)***
+2
+3
5/25 (20.0) 10/75 (13.3) 5/25 (20.0)*** 11/75 (14.7)a
3/25 (12.0) 2/25 (8.0)***
*Defined as sows presenting no corpora lutea and size of follicles £ 4 mm or none, in both ovaries. **Defined as sows presenting at least one cystic formation ‡ 15 mm and no corpora lutea, in any of their ovaries. ***Significantly different percentages ( p < 0.05). a p = 0.068.
The myometrium and/or endometrium of PRRSVinfected sows are edematous with lymphohistiocytic perivascular cuffs, and less commonly there are segmental lymphohistiocytic vasculitis in small vessels and microseperations between endometrial epithelium and trophoblasts (37). The histopathological results did not reveal lesions in the endometrium or myometrium in our study. On the contrary, in a previous study, multiple foci of lymphoplasmacytic inflammation were observed in the myometrium of sows after experimental infection with PRRSV (5). Furthermore, Lager and Halbur observed pleocellular endometritis and myometritis in uncomplicated PRRSV-induced abortion cases, suggesting that these uterine findings were also PRRSV induced (20). Moreover, myometritis, endometritis, and placentitis of the maternal placenta have been observed in experimentally and naturally infected sows (34). The design of this study was based on previous clinical observations at the slaughterhouse, which showed that the number of culled females from breeding stock due to ovarian cysts were higher in endemically PRRSV-infected farms when compared to other farms (31). The analysis of data of those farms showed that the main cause of early culling was fertility problems after weaning. In live animals, follicles maintained with a diameter of ‡ 2.0 cm for > 5 days after the onset of estrus have been regarded as cystic ovaries (4). Multiple large or small cysts without corpora lutea in the ovaries are common and are always associated with temporary or permanent infertility (16). Generally, in clinical practice, most sows with cystic ovaries are culled due to their failure to farrow or after repeat mating without conception. This problem leads to an increase in the nonproductive days of the breeding herd. Sows with ovarian cysts have been reported to have a higher incidence rate of return-to-estrus (34% vs. 7.7%) and anestrus sows that were not pregnant (10.6% vs. 0.6%), as well as a lower farrowing rate (52.2% vs. 90.0%) than normal sows (4). Most of the sows with multiple large cysts show intermittent or permanent anestrus. Previous studies have reported a link between reproductive problems and the presence of ovarian cysts found at the slaughterhouse (14,16,23,24). Additionally, ovarian cysts are a cause of lower conception rates, irregular estrous cycles, or behavioral changes, and they are present in approximately 10% of culled sows due to fertility problems (16,24). Generally, ovarian cysts in sows do not induce pathognomic clinical signs, other than an irregular and/or prolonged estrus cycle, permanent anestrus, and lower fer-
tility (4,17,32). In clinical practice, the use of gonadotropin PG600 (400 IU of equine chorionic gonadotropin and 200 IU of human chorionic gonadotropin) to anestrus sows that have not displayed estrus by day 7 post-weaning in PRRSV-infected farms could be a useful tool to increase estrus response in weaned sows (22). In addition, it is seems there is an essential relation between the presence of ovarian cysts and histological changes in the ovaries and oviductal and uterine mucosa, which eventually causes reproductive organ dysfunctions (36). If this state lasts for long, it may lead to permanent changes in the genital tract and result in persistent infertility of the sows, as well as an increase in the culling rate. The occurrence of luteinizing cysts in the present study was significantly lower in PRRSV-vaccinated animals ( p = 0.017) three semesters after the start of vaccinations and for the total trial period of 18 months ( p = 0.009). The results of the present study confirm the clinical observations of Papatsiros that ovarian cysts were more often observed in culled females from PRRSV endemic areas, as the occurrence of these luteinizing cysts were significantly decreased in PRRSV-vaccinated sows and gilts (31). The low female culling rate is an important management factor for the health status, economy, and production level of swine farms (19). In the case of PRRSV-infected farms, an increase of female culling rate occurs due to reproductive problems (30,39). A high removal rate requires a higher replacement rate, and this increases the number of nonproductive sow days, resulting in a higher pig production cost (7,8). Moreover, in a previous study of our group, the use of the same inactivated vaccine in sows and gilts proved to reduce the negative effects of PRRSV infection, improving significantly their reproductive performance and their litter characteristics (9). In addition, in another study, it is indicated that the same vaccination scheme of breeding stock leads to a significant reduction of culling rate due to reproductive failure 1.5 years after the start of vaccination (29). In conclusion, ovarian cysts are included in the causes of impaired fertility in endemic or chronic cases of PRRS. Based on the results of this study, PRRSV vaccination of breeding stock with an inactivated vaccine positively affects their reproductive system, as it is linked with a reduction in the occurrence of cystic formations (luteinizing cysts) in the ovaries (diameter > 2 cm). The results of this study are based on samples from a large-scale endemically PRRSVinfected farm. However, it would be interesting for future
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field trials to collect samples from PRRSV-vaccinated and nonvaccinated sows and replacement gilts from farms in different areas after initial infection or outbreak with PRRSV. The outcome of such follow-up studies would add valuable information on porcine health management that swine practitioners should apply in PRRSV-infected farms. Acknowledgments
This work was supported by Merial SAS through the Research Committee of the Aristotle University of Thessaloniki (Code no: 20567, Scientific Responsible S.C. Kyriakis). Author Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Dr. Vasileios G. Papatsiros Clinic of Medicine Faculty of Veterinary Medicine University of Thessaly 43100 Karditsa Greece E-mail: [email protected]
