RISK FACTORS ASSOCIATED WITH SWINE DYSENTERY IN EAST-EUROPEAN PIG PRODUCTION UNITS

The objective of the present study was to determine the risk factors for swine dysentery in East-European middle-size to large farrow to finish units, with separate breeding and grower-finisher facilities. Ten breeding animals (3-10% of the female inventory) and 10 grower finisher pigs (80-140 days of age) were sampled in each herd for polymerase chain reaction testing (PCR) for Brachyspira hyodysenteriae (B hyo) in their feces. Of 139 farrow to finish units, 51 (36.7%) were positive, 49 (35.3%) were negative, and 39 (28.1%) were inconclusive for B hyo by PCR. In breeding subunits, twelve variables passed the screening criterion for risk factors (P<.2) for B hyo PCR positivity. The odds of the breeding subunits being B hyo PCR positive were 3.5 times greater when the grower-finisher subunit was positive and the fiber content of the diet was >6%. Use of „all in all out” farrowing policy and having >60% multiparous sows, each reduced the odds of being B hyo PCR positive about fourfold. In growing-finishing subunits, fourteen variables passed the screening criterion for risk factors (P<.2) for B hyo PCR positivity. B hyo PCR positive status of the breeding subunits and higher fiber content of the diet were the most influential variable, with the odds of the grower-finisher subunits being B hyo PCR positive almost eight times greater when the breeding subunit was also B hyo PCR positive. Grower-finisher B hyo PCR positivity was also associated with the percentage of pigs housed on concrete slats, with the odds of being positive 7.5 times higher for subunits where more that 70% of the animals were kept on concrete slats compared to all other floor types. There was a strong association between grower-finisher status and whether

The objective of the present study was to determine the risk factors for swine dysentery in East-European middle-size to large farrow to finish units, with separate breeding and grower-finisher facilities.Ten breeding animals (3-10% of the female inventory) and 10 grower finisher pigs (80-140 days of age) were sampled in each herd for polymerase chain reaction testing (PCR) for Brachyspira hyodysenteriae (B hyo) in their feces.Of 139 farrow to finish units, 51 (36.7%) were positive, 49 (35.3%) were negative, and 39 (28.1%) were inconclusive for B hyo by PCR.In breeding subunits, twelve variables passed the screening criterion for risk factors (P<.2) for B hyo PCR positivity.The odds of the breeding subunits being B hyo PCR positive were 3.5 times greater when the grower-finisher subunit was positive and the fiber content of the diet was >6%.Use of "all in all out" farrowing policy and having >60% multiparous sows, each reduced the odds of being B hyo PCR positive about fourfold.
In growing-finishing subunits, fourteen variables passed the screening criterion for risk factors (P<.2) for B hyo PCR positivity.B hyo PCR positive status of the breeding subunits and higher fiber content of the diet were the most influential variable, with the odds of the grower-finisher subunits being B hyo PCR positive almost eight times greater when the breeding subunit was also B hyo PCR positive.Grower-finisher B hyo PCR positivity was also associated with the percentage of pigs housed on concrete slats, with the odds of being positive 7.5 times higher for subunits where more that 70% of the animals were kept on concrete slats compared to all other floor types.There was a strong association between grower-finisher status and whether

Introduction / Uvod
One hundred and thirty-nine East European farrow to finish "doublesite" production units (101--289 female inventory), clients of our consulting office, participated in the present survey.All units suffered sporadical occurrence of depressed weight gain and diarrhoea without blood.At necropsy watery green or yellow contents and adherent fibrinous exudate were present in the caecal and colonic mucosa.Histopathological examinations of secretions of the caecum and colon revealed moderate to severe typhlocolitis.Profiles of spirochetes were detected within the cytoplasma of enterocytes, between enterocytes or in macrophages in the lamina propria.Questionnaires have been used to collect detailed data about management, husbandry, mortality and culling rate (during the last 6 months and 3 years respectively), environmental and nutritional factors in these units.No clinical evaluations were performed.
Ten breeding animals (3-10% of the female inventory) of average parity (2.4+/-0.8 to 2.9+/-0.6SD) and average body condition (3.01+/-0.4 to 3.4+/ -05 SD) and 10 grower finisher pigs (80-140 days of age) were sampled in each herd for PCR testing of their feces.Rectal swabs were taken from randomly selected sows and the smallest looking pig in a grower--finisher pen.The swabs were immediately laid on ice and sent to the laboratory of our consulting office.In addition, intestinal spirochetes were isolated by anaerobic culture (37 o C) on sheep blood (SBA) selective colistin, vancomycin, and spectinomycin agar medium (CVS medium).Pure cultures of spirochetes were propagated either on SBA (incubated at 42 o C) in the Gas Pak Anaerobic System (BBL, Becton Dickinson Microbiology System) or in pre-reduced anaerobically-sterilised trypticase soy broth, as described by Kunkle et al.Š1986¹.Indole production, hyppurate hydrolysis, and the number of periplasmic flagella in porcine WBHIS (weakly b-hemolytic intestinal spirochaetes) were determined as described by Ramanathan et al.

Š1993¹.
Purified DNA was amplified by arbitrary-primed polymerase chain reaction (AP-PCR, Ralph, 1993) using the M13 reverse-sequencing primer.The amplified products were separated on 8% sodium dodecyl sulphate polyacrylamide gels and stained with silver nitrate and characterized using species--specific 23S-rDNA (rrl gene) sequences by PCR assays as stated by Leser et al. Š1997¹, andMuniappa et al. Š1997¹, and were diagnosed as positive, inconclusive or negative.
Statistical analyses were performed using BMDP 2D, 4F and LR (BMDP Statistical software, LA, CA, USA).Descriptive statistics were calculated for each variable, and continuous variables were categorized for use in analyses on the basis of the median and quartiles.For each potential risk factor, associations with B hyo status of the breeder and grower-finisher parts of the units were determined.Variables with P<.2 (chi-square) were considered for inclusion in multivariate logistic regression models.

Materials and methods / Materijal i metode rada
Separate logistic regression models were developed for the B hyo PCR status of breeder and grower-finisher parts of the units.Data for breeder and grower-finisher subunits of inconclusive B hyo status were excluded from the analysis to increase the herd-level-specificity of our classification without compromising herd-level-sensitivity as suggested by Jordan and McEwen Š1998¹, and Martin et al Š1991¹.Variables were added (stepwise inclusion) to the model when the chi-square P value for the variable was <.10.Interactions between factors in the final best-fitting models were also assessed.For the final models, adjusted odds ratio (OR) and 95% confidence intervals were obtained to quantify the strength of association with the different risk factors.The overall model fit was assessed using the H-L goodness-of-fit statistic as described by Hosmer and Lemshow Š1989¹.

PCR testing / PCR testiranje:
The median numbers of B hyo PCR positive samples were five (range 0 to 9) for the breeding subunits and two (rage zero to 9) for grower-finisher subunits.Of 139 farrow to finish units (including both, breeding and grower finisher subunits), 51 (36.7%) were positive, 49 (35.3%) were negative, and 39 (28.1%) were inconclusive for B hyo using our interpretation thresholds.Only the positive units were subjected to further evaluations.Risk factors for the B hyo PCR positivity in breeding subunits (table 1 and 2): Twelve variables passed the initial screening criterion for risk factors (P<.2) for B hyo PCR positivity.Management variables related to the grower-finisher unit were excluded from the analysis except for grower-finisher B hyo PCR status.The age when pigs left the breeding subunit and were transferred to the grower-finisher subunit has not been considered as a risk factor for the breeding units B hyo PCR status and was not included into the final model.
The odds of the breeding subunits being B hyo PCR positive and higher fiber content of the diet were 3.5 times greater when the grower-finisher subunit was positive and the fiber content of the diet was >6%.Use of "all in all out" farrowing policy and having >60% multiparous sows, each reduced the odds of being B hyo PCR positive about fourfold.Two-way interactions between risk factors were not significant (P<.05).The overall fit of the model was good (H-L goodness-of-fit c2=2.28 with 8df, P=.98).

Risk factors for the B hyo PCR positivity in grower-finisher subunits (table 3 and 4) / Faktori rizika za B hyo PCR pozitivnost u subjedinicama za zavr{nu fazu tova:
Fourteen variables passed the initial screening criterion for risk factors (P<.2) for B hyo PCR positivity.The breeding subunits B hyo PCR positive status was used as a surrogate variable to represent the combined effects of risk factors in the breeding subunits that might have influenced the risk of transmission of B Vet. glasnik 59 (3-4) 349 -362 (2005)  hyo to the grower-finisher subunits.The B hyo PCR positivity of the sows, fiber content of the diet, percentage of animals on concrete slats, rearing pigs on outdoor lots, and the number of pigs entering the subunits were included in the multivariable modeling.Diarrhoea, mortality, wasting, culling rate and culled because of diarrhoea were not further considered because it seemed that these were outcomes of B hyo caused SD, rather than risk factors for the disease.Similarly, due to the difficulty of interpreting categories such as treatment of sick animals or separation of them, were not included in the model.The B hyo PCR positive status of the breeding subunits and higher fiber content of the diet were the most influential variable, with the odds of the grower--finisher subunits being B hyo PCR positive almost eight times greater when the breeding subunit was also B hyo PCR positive.Grower-finisher B hyo PCR positivity was also associated with the percentage of pigs housed on concrete slats, with the odds of being positive 7.5 times higher for subunits where >70% of the animals were kept on concrete slats compared to all other floor types.There was a strong association between grower--finisher status and whether the animals were in outdoor lots, with the odds of being B hyo PCR positive substantially lower for pigs in outdoor lots compared with all other surfaces.Historically, observation of lesions at necropsy and histological examination have been the primary methods for diagnosis of SD ŠBilic and Bilkei, 2003¹.Nevertheless, there are economical and epidemiological limitations to postmortem examination of swine in the field ŠMauch and Bilkei, 2004¹.Necropsy is costly and it is not applicable to large scale screening for epidemiological investigations, and lesions may be resolved by the time of necropsy examination ŠBilic and Bilkei, 2003¹.Immunohistochemical or in situ hybridization with specific nucleic acid probes are highly specific, but lack sensitivity ŠDuhamel and Mathiesen, 1998¹.Consequently, antemortem diagnostic methods are to be used in the field.In the present study, it was difficult to determine the optimal cut-off value for classification of B hyo PCR positivity because of the lack of reliable estimates of the sensitivity and specificity of PCR for B hyo. Bilkei ŠMauch and Bilkei, 2004¹ suggested that the sensitivity of B hyo PCR for an individual test was approximately 90 and the specificity 99.However, because there is evidence that sensitivity varies with age ŠBilic and Bilkei, 2003¹, test sensitivity in field studies is often lower than in experimental studies.Further, under field conditions, there might be an increased risk of exposure to an organism that may induce crossreacting antibodies ŠGreiner and Gardner, 2000¹.Our solution to this problem was to require a minimum of three test--positive animals to designate a positive unit compared with the threshold of one or two B hyo PCR positive pigs that is commonly used in epidemiological studies.We excluded herds (subunits) with one or two B hyo PCR positive animals from the logistic models.Although this reduced the sample size for the analyses, we believe that this approach might have reduced possible misclassification of a herd's B hyo PCR status and resulted in less bias in the estimated odds ratios.
To our knowledge, there are no published studies of risk factors for B hyo infections in East--European middle sized to large indoor and outdoor pig herds.
The risk factors identified in this study were non-specific, with the exception of the use of concrete slats.Our cross sectional design of the study precluded us from differentiating factors associated with the introduction of B hyo and factors associated with transmission of the organism once infection had been estimated in a herd.
In the present study, we identified four factors (B hyo PCR status of the grower--finisher subunit, fiber content of the diet, farrowing house management, and parity of the breeding sows) that best explained the variation in PCR status in the breeding subunits.In the grower--finisher subunits five factors, as B hyo PCR status of the breeding subunit, fiber content of the diet, number of pigs that entered the grower--finisher subunit during the last 6 months, percentage of animals kept on concrete slats, and outdoor production) explain the variation in B hyo PCR status of the animals.In the present trial there was a strong association between the B hyo PCR status of the grower--finisher subunits and that of the breeding subunits.This finding suggests that B hyo is primarily transmitted by contact with infective fecal material and that the minimal infectious dose must be low ŠBaumann and Bilkei, 2002¹.In the present trial the farrowing barn and nursery of the breeding subunit may have acted as an important source for transmission of B hyo.The grower-finisher subunits may have had even greater potential for transmission of B hyo among pigs because of increased pig-to-pig contact due to high animal density, typical for grower-finisher production systems ŠBilkei, 1996b¹.
Environmental factors, including the design of the facility and management strategies, the level of hygiene, population density and the fiber content of the diet can affect the prevalence and severity of SD ŠBilkei, 1996b¹.In the present trial we found a greater risk of SD when pigs were kept on slats or meshed flooring and there was an association between housing on concrete slats and B hyo PCR positivity in nearly 70% of the cases.
The presented results indicate that a higher proportion of multiparous sows may result in lower transmission of B hyo.This might be explained either because a higher proportion of older sows may have a greater resistance to B hyo, or that in such cases a lower turnover of young breeding animals and less contact between susceptible gilts and multiparous sows diminishes the possibility of transmitting pathogens ŠBaumann and Bilkei, 2002¹.A lower turnover may reduce the introduction of pathogens as well.The all in all out farrowing policy suggests that such management reduces the transmission of pathogens by minimizing the direct and indirect contact between infected and suspected animals.
In the present study, we have not evaluated herds of more than 300 sows.In a study, smaller (<300) herd sizes, and all in all out production system were associated with lower occurrence of SD in East-European production units ŠMauch and Bilkei, 2004¹.
It is "widely believed" ŠBilic and Bilkei, 2003¹ that the clinical expression of swine dysentery might be influenced by diet.Prohaszka and Lukacs Š1984¹ suggested that by supplying more fermentable substrate to the colon, such a diet increases volatile fatty acid production, reducing colonic pH, which creates an unfavorable environment for the spirochetes.It has been stated ŠBilkei et al., 1995¹ that if the diet was changed to high fiber content, the bacterial buildup in the colon was decreased.In contrast to Bilkei et al. Š1995¹, Baumann and Bilkei Š2002¹ and Prohaszka and Lukacs Š1984¹, other authors ŠPluske et al., 1996;Siba et al., 1996¹ stated that highly digestible diets limit the amount of substrate entering the caecum and colon and therefore reduce microbial fermentation and volatile acid production, and concluded that highly digestible diets were protective against SD.Kirkwood et al.Š2000¹, failed to provide protection against SD with different diet formulations.Baumann and Bilkei Š2002¹ reduced gross pathological signs and economic losses feeding a high fermentable diet to pigs inoculated with B hyodysenteriae.The same authors evaluated the effect of highly fermentable fiber on the incidence and severity of swine dysentery (SD) after experimental oral infection with pure cultures of B. hyo.Pigs were fed until slaughter with a feed containing 9.6 % highly fermentable neutral detergent fiber or with 6.1% low fermentable neutral detergent fiber.Fecal shedding of B. hyo by PCR, antibody response by IFA, clinical signs, growth performance and extents of gross and microscopical lesions specific for SD were determined.Fecal shedding of B. hyo and antibodies specific for B. hyo were detected at day 30 post infection.Significant (p<0.05)milder clinical signs typical for SD were detected in the group fed with 9.6 % high fermentable fiber compared to animals fed with a feed containing 6.1% low fermentable neutral detergent fiber.Daily weight gain differed significantly (p<0.05) between the groups (group one 780 g vs. group two 760 g).Food conversion efficiency showed a significant (p<0.05)better (3.28) result in group one than in group two (3.38).Feed consumption presented significantly (p<0.001)better results in group one compared to group two (2.38 kg vs.

kg).
Conclusion, application of all in all out management in the farrowing and nursery facilities, B hyo negativity of adjacent grower-finisher units, high fiber content of the diet, and older parity structure in a sow herd may reduce the risk factors for SD.In grower-finisher units, slatted concrete flooring are associated with higher, while B hyo negativity of the breeding units, fiber content of the diet and outdoor production with lower risk of SD infections.
Nucleic acid-based diagnostic methods involving PCR amplification of DNA sequences specific for B hyo have been developed ŠLeser et al., 1997¹.Because the Vet.glasnik 59 (3-4) 349 -362 (2005) Mirko CP and Bilkei G: Risk factors associated with swine dysentery in East-European pig production units Discussion / Diskusija PCR assay can detect the presence of small numbers of B hyo directly in feces, it has been used as a tool to monitor the presence of B hyo in swine herds ŠLeser et al., 1997¹.PCR is a simple technique and can reliably detect B hyo, when grown from a primary isolation plate ŠAteyo et al., 1996¹.Definitive diagnosis of SD by culture alone is not possible, because several different intestinal spirochaetes can produce weak b-hemolysis when cultured anaerobically on selective blood agar media.We concluded that AP-PCR analyses of spirochetal DNA were reliable means for laboratory identification of SD-associated B. isolates.Hippurate hydrolysis only indicates the presence of B. pilosicoli, the other porcine Brachyspira spp, including B. hyo, are hippurate hydrolysis negative.