Antimicrobial susceptibility profiles of Mycoplasma hyorhinis strains isolated from five European countries between 2019 and 2021.

Mycoplasma hyorhinis is an emerging swine pathogen bacterium causing polyserositis and polyarthritis in weaners and finishers. The pathogen is distributed world-wide, generating significant economic losses. No commercially available vaccine is available in Europe. Therefore, besides improving the housing conditions for prevention, antimicrobial therapy of the diseased animals is the only option to control the infection. Our aim was to determine the minimal inhibitory concentrations (MIC) of ten antimicrobials potentially used against M. hyorhinis infection. The antibiotic susceptibility of 76 M. hyorhinis isolates from Belgium, Germany, Hungary, Italy and Poland collected between 2019 and 2021 was determined by broth micro-dilution method and mismatch amplification mutation assay (MAMA). Low concentrations of tiamulin (MIC90 0.312 µg/ml), doxycycline (MIC90 0.078 µg/ml), oxytetracycline (MIC90 0.25 µg/ml), florfenicol (MIC90 2 µg/ml) and moderate concentrations of enrofloxacin (MIC90 1.25 µg/ml) inhibited the growth of the isolates. For the tested macrolides and lincomycin, a bimodal MIC pattern was observed (MIC90 >64 µg/ml for lincomycin, tulathromycin, tylosin and tilmicosin and 5 µg/ml for tylvalosin). The results of the MAMA assay were in line with the conventional method with three exceptions. Based on our statistical analyses, significant differences in MIC values of tiamulin and doxycycline were observed between certain countries. Our results show various levels of antimicrobial susceptibility among M. hyorhinis isolates to the tested antibiotics. The data underline the importance of susceptibility monitoring on pan-European level and provides essential information for proper antibiotic choice in therapy.

Comparison of Primary Virus Isolation in Pulmonary Alveolar Macrophages and Four Different Continuous Cell Lines for Type 1 and Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15Sn-CD163, PK15S10-CD163). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15Sn-CD163, PK15S10-CD163, MARC-145Sn, and MARC-145. The titers in PK15Sn-CD163 and PK15S10-CD163 cells were significantly correlated with virus titers in PAM for both PRRSV1 (p < 0.001) and PRRSV2 (p < 0.001) compared with MARC-145Sn (PRRSV1: p = 0.22 and PRRSV2: p = 0.03) and MARC-145 (PRRSV1: p = 0.04 and PRRSV2: p = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145Sn cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future.

Implementation and evaluation of different eradication strategies for Brachyspira hyodysenteriae.

BACKGROUND: Brachyspira infections are causing major losses to the pig industry and lead to high antimicrobial use. Treatment of Brachyspira (B.) hyodysenteriae infections may be problematic due to the high level of antimicrobial resistance. The present study implemented and evaluated farm-specific eradication programmes for B. hyodysenteriae in 10 different infected pig farms in Belgium. RESULTS: Ten pig farms clinically infected with B. hyodysenteriae volunteered to implement a farm-specific eradication programme. The programme depended on the farm and management characteristics, antimicrobial susceptibility of the B. hyodysenteriae strain and the motivation of the farmer. Two farms practiced total depopulation, six farms partial depopulation and two farms antimicrobial medication without depopulation. In addition, all farms implemented biosecurity measures, and faeces samples were tested for the presence of B. hyodysenteriae at 6, 9 and 12 months after the start of the program. Single Brachyspira isolates from before and after the programme were typed using multilocus sequence typing (MLST).Eradication was successful in four farms. Two of them (farrow-to-finish and finishing herd) had applied total depopulation and respected a vacancy period of at least 3 weeks. A third farm (gilt farm) practised partial depopulation, the rooms remained empty for 28 days and changed the source of breeding gilts. The fourth farm practised partial depopulation, the stables remained empty for 3 weeks, and used antimicrobial medication. The eradication programme was not successful in six farms. Two of the latter farms only used medication without partial depopulation. Four farms practiced partial depopulation, one of them combined it with antimicrobial medication. The cleaning and disinfection procedures, rodent control, stand-empty period and/or other biosecurity measures in the six farms were not always implemented properly. In two of three farms, isolates belonging to the same MLST type were found before and after eradication. CONCLUSIONS: Total depopulation or partial depopulation combined with implementing strict biosecurity measures allowed eradication of B. hyodysenteriae from clinically infected pig farms. Programmes based on antimicrobials without depopulation or partial depopulation without strictly adhering to all suggested biosecurity measures were not successful. Stockmanship and motivation of the farmer to permanently maintain high biosecurity standards are essential for success.

Presence and mechanisms of acquired antimicrobial resistance in Belgian Brachyspira hyodysenteriae isolates belonging to different clonal complexes.

Swine dysentery (SD) is an economically important disease for which antimicrobial treatment still occupies an important place to control outbreaks. However, acquired antimicrobial resistance is increasingly observed in Brachyspira hyodysenteriae. In this study, the Minimal Inhibitory Concentrations (MIC) of six antimicrobial compounds for 30 recent Belgian B. hyodysenteriae isolates were determined using a broth microdilution method. In addition, relevant regions of the 16S rRNA, 23S rRNA and the L3 protein encoding genes were sequenced to reveal mutations associated with acquired resistance. Finally, a phylogeny was reconstructed using minimal spanning tree analysis of multi locus sequence typing of the isolates. For lincomycin, doxycycline, tylosin and tylvalosin, at least 70% of the isolates did not belong to the wild-type population and were considered to have acquired resistance. For valnemulin and tiamulin, this was over 50%. In all isolates with acquired resistance to doxycycline, the G1058C mutation was present in their 16S rRNA gene. All isolates showing acquired resistance to lincomycin and both macrolides displayed the A2058T mutation in their 23S rRNA gene. Other mutations in this gene and the N148S mutation in the L3 protein were present in both wild-type isolates and isolates considered to have acquired resistance. Multi locus sequence analysis revealed a previously undescribed clonal complex, with 4 novel sequence types in which the majority of isolates showed acquired resistance to all tested antimicrobial products. In conclusion, acquired antimicrobial resistance is widespread among Belgian B. hyodysenteriae isolates. The emergence of multi-resistant clonal complexes can pose a threat to swine industry.

Presence and characterization of pig group A and C rotaviruses in feces of Belgian diarrheic suckling piglets.

The importance of group A and C rotaviruses (RVA and RVC) in the pathogenesis of diarrhea in Belgian suckling pigs is poorly investigated, and it is not known which strains are circulating in the Belgian suckling pig population. Obtaining better insights in the occurrence of both viral species in the swine population is essential in order to develop accurate diagnostic, therapeutic and prophylactic strategies to protect suckling pigs against diarrhea in a durable manner. In the present study, viral loads of RVA and RVC were quantified in diarrhea samples of suckling piglets less than 2 weeks old, collected on 36 different Belgian farms. On 22 of 36 farms tested (61%), high viral loads of RVA (6.96-11.95 log10 copies/g feces) and/or RVC (5.40-11.63 log10 copies/g feces) were detected. Seventeen RVA isolates were genotyped for their outer capsid proteins VP7 and VP4. Four different G-genotypes (G3, G4, G5 and G9) for VP7 were found together with 4 different P-genotypes (P[6], P[7], P[13] and P[23]) for VP4, in 8 different G/P combinations. All characterized RVC strains belonged to genotype G6 (VP7), except for one strain possessing the G1 genotype. VP4 genes of Belgian RVC strains were genetically heterogeneous, but were classified in the genotype P5. Most rotavirus positive samples also contained Escherichia coli, whereas Clostridium perfringens infections were mainly detected in rotavirus negative samples. Results of the present study offer better insights in the occurrence of RVA and RVC infections in Belgian diarrheic suckling piglets. As a conclusion, routine diagnostic testing for both viral species in cases of diarrhea in suckling pigs is highly recommended. Furthermore, the present findings also offer valuable information for the development of new prophylactic measures against rotavirus. Finally, the relatedness between RVC strains from pigs and other host species is described, and their possible implications in interspecies transmission events are discussed.

Boar seminal plasma components and their relation with semen quality.

Select boar seminal plasma (SP) components and their relation to semen quality were investigated. Thirty nine boars from three artificial insemination (AI) centers were divided into group A (GA: > 80% normal sperm and >70% motility) and group B (GB: < 80% normal sperm and < 70% motility). Each ejaculate was collected and semen volume, concentration, sperm motility (computer aided semen analysis; CASA), morphology, and vitality (both eosin nigrosin staining) were investigated. The SP was separated and analyzed for aspartate-amino-transferase (AST), γ-glutamyl-transferase (GGT), alkaline phosphatase (ALP) activity, and the concentrations of sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphate (PO(4)(3-)), magnesium (Mg), selenium (Se) and zinc (Zn) were assessed. Repeated measures (2 months interval) were conducted in eight boars of GA from one AI center. The activity of GGT (r = -0.482) and ALP (r = -0.459) was moderately associated (p < 0.05) with ejaculate volume and strongly associated with concentration (r = 0.580 and r = 0.618, respectively; p = 0.000). Moderate associations (p < 0.05) were found between ALP (r = 0.439), GGT (r = 0.387), Na (r = -0.428), K (r = 0.354), and Se (r = 0.354) with progressive motility. The SP concentration of Na (r = -0.401), Cl (r = -0.521), and K (r = 0.350) was associated (p < 0.05) with normal morphology. Only Mg was associated (p < 0.05) with membrane damage (r = -0.335). The concentration of Na, Cl, and Zn (1681.0 vs. 1701.0 µg/dL) was different between groups (p < 0.05). Repeated measures showed significant differences in time but only for Na, Mg, and Zn (p < 0.05). In conclusion, several biochemical components of SP were related to semen quality. The analysis of biochemical parameters could provide extra information about reproductive health of AI boars.

Tick-borne encephalitis virus seropositive dog detected in Belgium: screening of the canine population as sentinels for public health.

Tick-borne encephalitis virus (TBEV) is an important emerging tick-borne viral infection of humans and dogs in Europe. Currently, TBEV surveillance is virtually nonexistent in Belgium, which is considered nonendemic. A commercial enzyme-linked immunosorbent assay (ELISA) was adapted for the detection of TBEV-specific IgG-antibodies in canine sera. Serum samples of Belgian dogs were obtained from three diagnostic laboratories from Northern (n=688) and Southern Belgium (n=192). ELISA-positive and borderline samples were subjected to a TBEV rapid fluorescent focus inhibition confirmation test. One dog was confirmed TBEV seropositive. Several ELISA-positive and borderline sera underwent seroneutralization and hemagglutinin inhibition tests to rule out West Nile and Louping Ill viruses, but tested negative. The clinical history of the seropositive dog could not explain beyond doubt where and when TBEV infection was acquired. Further surveillance is necessary to determine whether this dog remains a single travel-related case or whether it represents an early warning of a possible future emergence of TBEV.

Effectiveness of the sperm quality analyzer (SQA-Vp) for porcine semen analysis.

The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of porcine semen. Both fresh and diluted semen from 50 different boars from a commercial artificial insemination (AI) centre were investigated. For the fresh ejaculate, the concentration obtained with SQA-Vp was compared with a photometer and a haemocytometer. For the diluted samples, the concentration and motility were compared with computer assisted semen analysis (CASA) and visual sperm analysis. The agreement between methods was studied with Bland-Altman plots and the repeatability with coefficient of variation (CV) as well as Bland-Altman plots. The sperm concentration (x10(6)/ml) obtained with SQA-Vp (379.3 ± 134.9) for fresh ejaculates agreed well with concentration by the photometer (447.2 ± 154.2; difference= -67.9 x 10(6)/ml; difference + 2SD = 55.3 x 10(6)/ml; difference - 2SD = -191.1 x 10(6)/ml) and with the haemocytometer (332.8 ± 141.11; d = 92.8; d + 2SD = 448.6; d - 2SD = -263). For diluted semen, the agreement between the concentration (x10(6)/ml) assessed with SQA-Vp (20.4 ± 4.3) was good with CASA (23.2 ± 5.8; d = -2.8; d + 2SD = 6.2; d - 2 SD = -11.8) but poor with the haemocytometer (18.8 ± 5.0; d = 1.6; d+ 2SD = 12.2; d - 2SD = -9). The % motile spermatozoa assessed by SQA-Vp (65.8 ± 10.0) in diluted semen agreed well with CASA (72.2 ± 13.7; d = -6.4; d+ 2SD = 20; d - 2SD = -32.8) and with visual assessment (64.1 ± 11.6; d = 1.7; d+ 2SD = 30.9; d - 2SD = -27.5). The SQA-Vp showed a good repeatability (CV; repeatability coefficient) for measuring the concentration of both fresh (3.9%; d = 10.7; d + 2SD = 30.9; d - 2SD = -9.5) and diluted semen (2.6%; d = 1.0; d + 2SD = 2.38; d - 2SD = -0.42) and for motility (3.2%; d = 0.9; d + 2SD = 8.5; d - 2SD = -6.7). The mean SQA-Vp values fell between the other methods' results for both fresh and diluted semen. Moreover the repeatability was acceptable. Therefore SQA-Vp can be used as a valid device for sperm quality analysis in pigs.

Effects of toltrazuril on the growth of piglets in herds without clinical isosporosis.

Isospora suis is a widely prevalent and economically important parasite. The antiprotozoal compound, toltrazuril, was given as a single treatment to piglets without clinical signs in 10 herds. The daily weight gain (DWG) and mortality between the treatment date and weaning was compared on each herd in 10 treated and 10 control litters. The faeces of control litters were examined for oocyst excretion. In six herds, no oocysts were detected and treatment had no effect on DWG or mortality. In four pig herds, oocysts were detected and toltrazuril treatment significantly improved DWG by 25 g (P=0.003). Mortality rate was not affected. The partial economic benefit of toltrazuril treatment in positive herds was 0.20 euros per piglet. A single toltrazuril treatment of piglets in herds without clinical signs of isosporosis but with oocysts detected significantly improved DWG and was considered economically justified on each farm.

Transmission of encephalomyocarditis virus in pigs estimated from field data in Belgium by means of R0.

Transmission of encephalomyocarditis-virus (EMCV) has been estimated in experiments, but never using field data. In this field study, a farm in Belgium was selected where the presence of EMCV was confirmed by necropsy and virus isolation. Serology was used to estimate the transmission parameter R0. In one compartment with 630 pigs, 6 pens were fully sampled, in the remaining 38 pens, 2 randomly selected pigs were bled. The 151 pigs were bled twice and their serum was tested in a virus neutralisation test. Seroprevalence at the first and second sampling was 41 and 43% respectively, with a cut off value of 1:40. R0 was estimated for 2 scenarios, in- and excluding mortality based on the final sizes from the serological results of the second sampling. The R0 for the fully sampled pens was estimated between 0.6 and 1.7, the combined estimated R0 of these 6 pens was 1.36 (95%-CI 0.93-2.23). The median of the estimated R0 of the partially sampled pens was 1.3 and 1.4. Sampling two pigs per pen provided insight into the spread of the virus in the compartment, while the fully sampled pens provided an accurate estimation of R0. The low R0 strongly suggests that EMCV is not very effectively transmitted between pigs. The number of seropositive pigs in a pen and the spread in the compartment suggests that other routes of infection are more important, in this case most likely rodents. Preventing viral spread should therefore be focussed on rodent control instead of reduction of contact between pigs.