Slov Vet Res 2017: 54 (4):149-54
DOI: 10.26873/SVR-360-2017
UDC 636.4.09:616-036.22:579.841: 57.083.1
Original Research Article
SWINE BRUCELLOSIS CAUSED BY Brucella suis BIOVAR 2 IN CROATIA
Zeljko Cvetnic1, Sanja Duvnjak1, Maja Zdelar-Tuk1, Irena Reil1, Marina Mikulic2, Marija Cvetnic3, Silvio Spicic1*
1Department of Bacteriology and Parasitology, 2Department of Veterinary Public Health, Croatian Veterinary Institute, Savska cesta 143, 3Faculty of Veterinary Medicine, University of Zagreb, Heinzelova cesta 55, Zagreb, Croatia
Corresponding author, E-mail: spicic@veinst.hr
Abstract: Brucellosis in swine was surveyed from 2011 to 2015 in 13 counties in Croatia. A total of 3230 breeding males were tested serologically, and positive reactions were confirmed in 42 (1.3%) males from 17 farms. A total of 641 sows with abortion or reproductive problems were tested, and positive reactions were confirmed in 34 (5.3%). Organs from 68 swine were tested for bacteria, and Brucella spp. was isolated from 47 (69.1%). B. suiswas identified in 45 isolates from domestic swine and 2 isolates from wild boar in six counties in Croatia, and all isolates were found to be B. suis biovar 2 based on Bru-up/Bru-low, Bruce-ladder, Suis-lad-der and RFLP-based PCR assays. These results indicate that brucellosis is difficult to eradicate in free-range and semi-free-range swine farming, particularly in areas where contact with wild boar is possible. Further disease control measures are required.
Key words: Brucella suls biovar 2; swine; abortion; prevalence; Croatia
Introduction
Brucella suis appears in most countries containing domestic swine and wild boar. B. suis infections spread easily on swine farms and are difficult to control; the most common clinical signs are abortions and infertility in sows, mortality of offspring and orchitis in breeding males (1). B. suis biovars (bv.) 1, 2 and 3 cause brucellosis in swine. B. suis bv. 1 has been reported in Latin America (2, 3) as well as in USA and China (4, 5).
Received: 27 January 2017 Accepted for publication: 13 April 2017
B. suis bv.3 has also been detected in the USA and China (4, 5). B.suis bv. 2 is the most frequent cause of infection in domestic swine in Central and Western Europe, where wild boar and hares serve as natural carriers (6-12).
In the Central European country of Croatia, infection of horses and swine with B. suis bv. 3 has been reported based on classical microbiological assays (13, 14). Genotyping of B. suis in Croatia based on multi-locus, variable-number tandem repeat analysis revealed the existence of various B. suis strains with more or less different geographic distributions (22); some of the strains were identical to ones identified in Hungary, Germany
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and France. One of the drivers of B. suis infection in Croatia appears to be extensive domestic swine-holding under conditions in which contact or even natural mating with infected wild boar is possible, which has already been reported in other European countries (14-21).
To gain additional insights into the epidemiology of brucellosis, the B. suis biovar(s) responsible and the factors that may drive B. suis infection in Croatia, we surveyed large number of breeding swine males and sows from herds with abortions and reproductive problems from 13 counties in the country. Surveyed swine were free-range or maintained under semi-intensive conditions.
Material and methods
Description of the sample
In Croatia, any abortion or appearance of clinical signs in breeding swine that raises suspicions of brucellosis must be reported to a veterinarian in order to facilitate early detection. In these cases, an authorised veterinarian must take appropriate samples and submit them to an authorised brucellosis testing laboratory. Young boars must also be serologically tested for brucellosis prior to their use in breeding programs, artificial insemination, or natural mating.
Between 2011 and 2015, swine were surveyed in free or extensive rearing systems in which abortion had appeared in gravid sows, or reproductive problems such as infertility, stillbirths or failure to fertilise. Most of these systems were small farms with a few sows and young breeding males, which were held extensively, left to roam freely in the forest or kept free-range under natural conditions. At each farm where brucellosis was confirmed in sows, all breeding males were tested serologically. When young boar tested positive, testing was also performed on the swine farms where the boar were used to fertilise sows. All serologically positive swine were removed from breeding and sampled for bacteriology at the time of slaughter. These samples were kept at 4°C andtested within 24 h.
Serological examination
Serum samples. Blood samples were collected from 641 sows from 62 herds as well as from3230 boars from the following 13 counties in Croa-
tia:Bjelovar-Bilogora, Brod-Posavina, Karlovac, Koprivnica-Krizevci, Krapina-Zagorje, Medimurje, Osijek-Baranja, Pozega-Slavonia, Sisak-Moslavi-na, Varazdin, Virovitica-Podravina, Vukovar-Sri-jem, and Zagreb County.
Serological tests. Serum was assayed using the Rose Bengal test (RBT), complement fixation test (CFT), and the INgezim Brucella Porcina kit (Ingenasa, Madrid, Spain), which is an indirect enzyme-linked immunosorbent assay. Antigens for RBT and CFT were produced by the Institut Pourquier (Montpellier, France) and the Croatian Veterinary Institute (Zagreb, Croatia).Tests were conducted according to OIE guidelines(23, 24), while the INgezim kit was used according to the manufacturer's recommendations.
Bacteriological examination
Tissue samples. A total of 150 samples were collected at slaughter from 68 domestic sows, hogs and other swine from six counties. Samples comprised lymph nodes (parotid, submandibular, retropharyngeal, portal, subiliac, mesothelial, supramammary) (n = 62), spleen (8), testicles (18), foetuses (12) and uterus (50).
Bacteriological tests. Tissue samples were homogenised in a stomacher, and the suspension was directly cultured in duplicate on blood agar, Brucella agar and Farrell medium. One set of three plates was incubated at 37°C in a normal atmosphere, while the other set was incubated in a 10% CO2 atmosphere. Colony growth and morphology was monitored daily, and colonies were subcultured and examined by microscopy. Isolates were confirmed as Brucella using classical microbiological biotyping based on microscopy, culture and biochemistry (23-25).
Polymerase chain reaction (PCR)-based
biotyping
Isolates were confirmed to be Brucella using Brucella genus-specific PCR (26). The reference method to confirm Brucella species was Bruce-ladder multiplex PCR (27), while Suis-ladder multiplex PCR was used to determine B. suis biovars (28).
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Table 1: Number of blood samples tested from pigs and wild boars
Year	Sows tested (n)	Breedings (n)	Positive sows (n/%)	Tested swine (n) -positive farms* (n/%)	Positive farms (n/%)	Wild boars tested (n)	Positive wild boars (n / %)	Positive hunting areas (n)
2011	211	32	10 / 4.7	511 - 27 / 5.3	4 / 12.5	1129	11 / 0.97	5
2012	170	19	7 / 4.1	314 - 12 / 3.8	2 / 10.5	896	4 / 0.44	2
2013	97	6	5 / 5.2	272 - 15 / 5.5	2 / 33.3	425	8 / 1.88	5
2014	116	3	9 / 7.8	392 - 10 / 2.6	1 / 33.3	445	3 / 0.67	1
2015	47	2	3 / 6.4	257 - 3 / 1.2	1 / 50.0	335	8 / 2.39	4
TOTAL 641	62 34 / 5.3 1746 - 67 / 3.8 10 / 16.1 3230 42 / 1.3 17
Results
Serological examination
Between 2011 and 2015, 3230 breeding males from 13 Croatian counties were serologically analysed, and positive reactions were confirmed in 42 (1.3%) boars from five counties (Table 1). Over the same period, 641 sows that aborted or displayed reproductive problems were serologically tested, and positive reactions were confirmed in 34 (5.3%). On farms containing sows positive for brucellosis, all other swine were serologically analysed, and positive reactions were found in 67 (3.8%) of swine on 10 farms in the same five counties (Table 1).
Bacteriological examination
Organs of 68 swine were tested bacteriologically, and B. suis bv. 2 was identified in 45 domestic swine (66.2%) from five counties (Bjelovar-Bilogora, Virovitica-Podravina, Sisak-Moslavina, Brod-Posavina and Zagreb) and 2 wild boars (2.9%) from Zagreb and Vukovar-Srijem counties.
PCR assay
A total of 47 B. suis isolates were typed using the Bru-up/Bru-low and Bruce-ladder PCR assays to identify genus and species, respectively, as well as the Suis-ladder PCR assay to assign biovar. Based on reference samples, all isolates were identified as B.suis bv. 2.
Discussion
This survey of a relatively large swine population from around Croatia confirms and extends previous findings that brucellosis poses a threat on small farms that share breeding males and in systems where swine are kept extensively or freerange at pasture and where contact with wild boar is possible. Several studies indicate that swine on farms typically become infected following the introduction of infected sows or breeding males, or through contact with infected wild boar(1, 14, 19, 21). Our findings of B. suis bv. 2 in 45 domestic swine and 2 wild boar from six Croatian counties highlights the difficulty of eradicating brucellosis from swine populations held semi-intensively or allowed to roam freely at pasture.
This survey is consistent with several earlier studies of Croatian countries bordering the Sava River, which identified B. suis bv. 2 as the cause of brucellosis in domestic swine and wild boar. In these counties, breeding swine are often held extensively at pasture or in forests, where contact is possible with many other swine as well as wild boar (8, 9, 14, 18, 19). Our findings are also consistent with studies in several Western European countries. Swine brucellosis caused primarily by B. suis bv. 2 has been reported in Austria, Germany, Portugal and Spain. A study of 36 swine herds in Sardinia found 33% to be positive, with the infecting strain in all cases being B. suis bv. 2 (1), and a study of 28 sows with reproductive problems in the Rome area found 89% to have brucellosis, with the infecting strain
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being B. suis bv. 2 (21). However, B. suis infections have yet to be reported in Finland, Sweden, UK or Norway, and they have not been reported in Belgium since 1969 or in the Netherlands since 1973 (6).
The present survey detected B. suis bv. 2 in two wild boar, consistent with earlier reports of persistent B. suis bv.2 infection of wild boar in multiple regions of Croatia (8, 9, 14, 19). B. suis bv. 2 has been isolated from wild boar in many Central and Western European countries, including France (7, 15), Switzerland (16, 29), Germany (30), Belgium (11), Spain (17, 31) and Italy (12). Though direct evidence is lacking, it seems extremely likely that wild boars are a reservoir and source of infection for domestic swine. The two animal populations inhabit the same areas in nature and therefore indirect and direct (sexual) contact is possible.
Understanding B. suis epidemiology is important not only for the swine industry but also for other types of animal production, since the bacterium can spread easily from swine to other species. B. suis bv. 2 infection of dairy cows has been reported in Poland and Belgium (32, 33), and B. suis bv. 2 infection of roe deer (Capreolus capreolus) has been reported in Germany (34). B. suis infection of dogs used to hunt wild boar has been reported in the USA (35), and B. suis bv. 1 infection of armadillos (Chaetophractus villosus) has been described in Argentina (36). One report described B. suis infection of horses in Croatia (13), and while those authors identified the strain as bv. 3 based on biochemical assays, subsequent analysis of single-nucleotide polymorphisms suggest it maybe bv. 1 (37), while the observed zoonotic potency suggests it may be bv. 2 or perhaps a novel strain (18).
Previous work suggests that brucellosis is not a widespread problem among swine populations raised in intensive rearing conditions or on large farms with semi-intensive rearing (18, 19), which were covered in the present survey also.
The Croatian counties where the present survey detected swine brucellosis share long borders with several European countries: Slovenia, Hungary, Serbia, and Bosnia and Herzegovina. In this way, B. suis bv. 2 poses a regional threat for brucellosis control, which should be addressed through strong early-detection programs and rapid response in the event of confirmed cases.
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BRUCELOZA PRAŠIČEV, POVZROČENA Z BAKTERIJO Brucella suis BIOVAR 2 NA HRVAŠKEM
Ž. Cvetnic, S. Duvnjak, M. Zdelar-Tuk, I. Reil, M. Mikulic, M. Cvetnic, S. Špičic
Povzetek: Brucelozo pri prašičih smo spremljali od leta 2011 do leta 2015 v 13 hrvaških občinah. S serološko analizo smo preverili prisotnost bruceloze pri 3230 samcih v razplodu. Pozitivne reakcije smo ugotovili pri skupno 42 merjascih s 17 farm, kar predstavlja 1,3 % živali. S serološkimi testi smo preverili prisotnost protiteles proti bruceli tudi pri 641 plemenskih svinjah, ki so zvrgle ali imele težave z zabrejitvijo. Pozitivna reakcija je bila ugotovljena pri 34 svinjah, kar predstavlja 5,3 % vseh testiranih živali. Notranje organe 68 svinj iz šestih občin smo uporabili za osamitev bakterij Brucellaspp. Bakterije smo ugotovili pri 47 vzorcih (69,1 %). Bakterijo Brucella sulssmo odkrili v vseh 47 vzorcih, izmed katerih jih je bilo 45 od domačih plemenskih svinj, dva vzorca pa sta bila od divjih svinj. Vse izolirane bakterije so pripadale sevu B. suls biovar 2, kot so pokazale dodatne analize z uporabo metod Bru-up/Bru-low, Bruce-ladder, Suis-ladder in RFLP. Ti rezultati kažejo, da je popolno izkoreninjenje bruceloze težavno, še posebej v prostih rejah prašičev, kjer obstajajo možnosti stika z divjimi prašiči. Zato bi bilo v prihodnje potrebno razmisliti o dodatnih načinih nadzora nad to nevarno boleznijo prašičev.
Ključne besede: Brucella suls biovar 2; prašiči; zvrg; pojavnost; Hrvaška