Volume 66 (2022): Issue 1 (March 2022)

Highly pathogenic avian influenza (HPAI) outbreaks caused by the Gs/Gd lineage of H5Nx viruses occur in Poland with increased frequency. The article provides an update on the HPAI situation in the 2020/2021 season and studies the possible factors that caused the exceptionally fast spread of the virus.

Samples from poultry and wild birds delivered for HPAI diagnosis were tested by real-time RT-PCR and a representative number of detected viruses were submitted for partial or full-genome characterisation. Information yielded by veterinary inspection was used for descriptive analysis of the epidemiological situation.

The scale of the epidemic in the 2020/2021 season was unprecedented in terms of duration (November 2020–August 2021), number of outbreaks in poultry (n = 357), wild bird events (n = 92) and total number of affected domestic birds (approximately ~14 million). The major drivers of the virus spread were the harsh winter conditions in February 2020 followed by the introduction of the virus to high-density poultry areas in March 2021. All tested viruses belonged to H5 clade 2.3.4.4b with significant intra-clade diversity and in some cases clearly distinguished clusters.

The HPAI epidemic in 2020/2021 in Poland struck with unprecedented force. The conventional control measures may have limited effectiveness to break the transmission chain in areas with high concentrations of poultry.

The lack of proofreading activity of the viral polymerase and the segmented nature of the influenza A virus (IAV) genome are responsible for the genetic diversity of IAVs and for their ability to adapt to a new host. We tried to adapt avian IAV (avIAV) to the pig by serial passages in vivo and assessed the occurrence of point mutations and their influence on viral fitness in the pig’s body.

A total of 25 in vivo avIAV passages of the A/duck/Bavaria/77 strain were performed by inoculation of 50 piglets, and after predetermined numbers of passages 20 uninoculated piglets were exposed to the virus through contact with inoculated animals. Clinical signs of swine influenza were assessed daily. Nasal swabs and lung tissue were used to detect IAV RNA by real-time RT-PCR and isolates from selected passages were sequenced.

Apart from a rise in rectal temperature and a sporadic cough, no typical clinical signs were observed in infected pigs. The original strain required 20 passages to improve its replication ability noticeably. A total of 29 amino-acid substitutions were identified. Eighteen of them were detected in the first sequenced isolate, of which 16 were also in all other analysed strains. Additional mutations were detected with more passages. One substitution, threonine (T) 135 to serine (S) in neuraminidase (NA), was only detected in an IAV isolate from a contact-exposed piglet.

Passaging 25 times allowed us to obtain a partially swine-adapted IAV. The improvement in isolate replication ability was most likely related to S654 to glycine (G) substitution in the basic protein (PB) 1 as well as to aspartic acid (D) 701 to asparagine (N) and arginine (R) 477 to G in PB2, glutamic acid (E) 204 to D and G239E in haemagglutinin and T135S in NA.

African swine fever virus (ASFV) causes one of the most dangerous diseases of pigs and wild boar – African swine fever (ASF). Since its second introduction into Europe (in 2007), the disease has been spreading consistently, and now ASF-free European countries are at risk. Complex interactions between the host’s immune system and the virus have long prevented the development of a safe vaccine against ASF. This study analysed the possibility of neutralisation of the ASFV in vitro by sera collected from ASF-survivor animals.

Two pig and three wild boar serum samples were collected from previously selected potential ASF survivors. All sera presented high antibody titres (>5 log₁₀/mL). Primary alveolar macrophages were cultured in growth medium containing 10% and 20% concentrations of selected sera and infected with a haemadsorbing ASFV strain (Pol18_28298_O111, genotype II). The progress of infection was investigated under a light microscope by observing the cytopathic effect (CPE) and the haemadsorption phenomenon. Growth kinetics were investigated using a real-time PCR assay.

Haemadsorption inhibition was detected in the presence of almost all selected sera; however, the inhibition of virus replication in vitro was excluded. In all samples, a CPE and decreasing quantification cycle values of the viral DNA were found.

Anti-ASFV antibodies alone are not able to inhibit virus replication. Interactions between the humoral and cellular immune response which effectively combat the disease are implicated in an ASF-survivor’s organism.

Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims.

Fifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus’ genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the ORF2 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established.

The results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R²) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%).

This study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4.

Akabane virus (AKAV) has been detected in a variety of host species in China, but there are only limited records of its occurrence in goats. However, more attention needs to be paid to understanding the diversity of viruses in this species. The aim of the study was to explore the genotype characteristics and variation trend of AKAV and their relationship with virulence in Yunnan, China.

Blood samples were collected from goats during routine surveillance of goat diseases in Yunnan province in 2019. The AKAV CX-01 strain was isolated using BHK-21 cells. To understand pathogenicity, the virus was intraperitoneally (IP) and intracerebrally (IC) inoculated into suckling mice and tissue samples were subsequently analysed histopathologically and immunohistochemically.

Akabane virus CX-01 strain induced encephalitis and impairment of the central nervous system with fatal consequences. Phylogenetic analysis based on the ORF sequences of the small segments indicated that the AKAV isolate used was most closely related to the GD18134/2018 Chinese midge and bovine NM BS/1strains, while phylogenetic analysis based on the medium segments showed a close relationship between CX-01 and the Chinese GLXCH01 strain.

The CX-01 isolate was related to AKAV genogroup Ia and probably originated from a recombination of different strains.

Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection.

The gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA.

Positive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal.

The established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies.

The study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current ante-mortem testing protocols (the tuberculin skin and Enferplex Camelid TB tests) to identify TB-free alpaca herds and individuals for export. Our research and the available literature indicate that the alpaca (Vicugna pacos) is extremely susceptible to Mycobacterium bovis infection, and that testing periodicity fails to take into account that animals do not manifest disease symptoms for a long time. The skin test failed to identify Mycobacterium bovis infection in two alpacas prior to their movement from the UK to Poland. The animals were purchased by a breeding centre in Poland, and were then shown at an international animal exhibition. The last owner of the alpacas before their deaths from TB bought the infected animals unwittingly in order to run rehabilitation activities with disabled children on his farm.

Thoracic lymph node, lung and liver tissue samples obtained at necropsy were examined histopathologically after Ziehl–Neelsen staining. Tissue samples were homogenised and mycobacteria present there were cultured on Stonebrink’s medium during a 6-week incubation. A commercial test using polymorphism of the chromosomal direct repeat region provided species identification and additional identification was by spacer oligonucleotide typing and mycobacteria interspersed repetitive unit–variable number tandem repeat analysis with a gel electrophoresis protocol.

The microbiological examination confirmed multiorgan TB caused by the SB0666 spoligotype of Mycobacterium bovis.

Due to the suboptimal performance of current diagnostic tests for TB in alpacas, there is a risk that infected animals may be moved unwittingly. A risk of TB spread associated with the international movement of alpacas is implied by this study.

Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP), causes economic losses in dairy herds due to reduced milk production and premature culling. A test-and-cull strategy coupled with changes in calf rearing management preventing new infections has been introduced into infected herds to control MAP prevalence. This study appraised the effectiveness of these practice changes.

In 19 large dairy herds (of a median 470 milk-producing cows), implementing MAP control measures for 3–7 years, a serum ELISA was used to detect infected cows in their dry-off period. The number of ELISA-positive animals per year (EPAY) was calculated and statistical analysis was used to test whether the EPAY total decreased during the control period and to analyse the EPAY in relationship to the duration of the control programme.

Statistical support was found for a decrease of EPAY over time (P < 0.01, odds ratio 0.756) and in 14 herds a significant fall in the percentages of EPAY during the test period (P ≤ 0.05) was noted.

Our results demonstrated the effectiveness of the control measures in place to reduce MAP infection in herds with initial EPAY ≥3.36%. The missing decreasing trend in the remaining five herds with low average initial EPAY suggested the need for additional measures to reduce the number of infected animals in these herds.

Nontuberculous mycobacteria (NTM) are increasingly recognised as causative agents of opportunistic infections in humans for which effective treatment is challenging. There is very little information on the prevalence of NTM drug resistance in Poland. This study was aimed to evaluate the susceptibility to antibiotics of NTM, originally isolated from diseased ornamental fish.

A total of 99 isolates were studied, 50 of them rapidly growing mycobacteria (RGM) (among which three-quarters were Mycobacterium chelonae, M. peregrinum, and M. fortuitum and the rest M. neoaurum, M. septicum, M. abscessus, M. mucogenicum, M. salmoniphilum, M saopaulense, and M. senegalense). The other 49 were slowly growing mycobacteria (SGM) isolates (among which only one was M. szulgai and the bulk M. marinum and M. gordonae). Minimum inhibitory concentrations for amikacin (AMK), kanamycin (KAN), tobramycin (TOB), doxycycline (DOX), ciprofloxacin (CIP), clarithromycin (CLR), sulfamethoxazole (SMX), isoniazid (INH) and rifampicin (RMP) were determined.

The majority of the isolates were susceptible to KAN (95.95%: RGM 46.46% and SGM 49.49%), AMK (94.94%: RGM 45.45% and SGM 49.49%), CLR (83.83%: RGM 36.36% and SGM 47.47%), SMX (79.79%: RGM 30.30% and SMG 49.49%), CIP (65.65%: RGM 24.24% and SGM 41.41%), and DOX (55.55%: RGM 9.06% and SGM 46.46%). The majority were resistant to INH (98.98%: RGM 50.50% and SGM 48.48%) and RMP (96.96%: RGM 50.50% and SGM 46.46%).

The drug sensitivity of NTM varies from species to species. KAN, AMK, CLR and SMX were the most active against RGM isolates, and these same four plus DOX and CIP were the best drugs against SGM isolates.

Ornithobacterium rhinotracheale (ORT) causes significant economic losses to the poultry industry around the world. The bacterium often affects poultry as part of multiple infections causing very serious clinical signs that are usually not limited only to the respiratory system. This study’s main objective was the retrospective detection and identification of ORT in turkey flocks.

ORT identification was performed in 6,225 samples taken from 133 different flocks between 2015 and 2020. Molecular methods were used, specifically real-time PCR and traditional PCR. We focused on partial 16S rRNA gene sequences of isolates, which were compared with sequences obtained from GenBank. The reaction products were analysed phylogenetically. Molecular methods indicating secondary infections was carried out, and the bacterial composition of the upper respiratory tract was 16S metasequenced for selected flocks to identify any other pathogens.

The presence of ORT was detected in 30.83% of samples by real-time PCR and 28.57% by PCR. Phylogenetic analysis of the PCR products from the turkeys samples showed that their sequences resolved into two main genetic groups. Tests for the occurrence of secondary infections showed the presence of Mycoplasma gallisepticum and M. synoviae in some samples but the total absence of Bordetella avium. The upper respiratory tract in turkeys was dominated by two major phyla Firmicutes and Proteobacteria. At the genus level, the genera Ornithobacterium, Mycoplasma, Gallibacterium, Avibacterium, and Escherichia-Shigella were found which may include pathogenic bacteria that can cause clinical symptoms.

The results of the analysis of multiple infection carried out in flocks with respiratory signs are probably associated with outbreaks of ornithobacteriosis in turkey flocks in Poland.

Domestic poultry is a natural reservoir of Campylobacter, the host–pathogen interaction being predominantly asymptomatic. This study investigated whether chickens remain asymptomatic partly because of lactic acid bacteria (LAB).

Campylobacter spp. and LAB were isolated from the gut of poultry chickens using enrichment and screening assays and were identified via rDNA sequencing. The C. jejuni DC3 isolate was grown in different cell-free supernatants (CFS) generated from a priority LAB isolate. An in vivo challenge involving the C. jejuni and LAB isolates using a chicken model was performed to confirm the in vitro findings.

Twelve presumptive LAB isolates had anti-C. jejuni activity based on cross-streak and agar plug assays, with Lactobacillus sakei L14 isolate exhibiting the highest activity. Inhibition by L. sakei L14 CFS of the growth of C. jejuni occurred in a dose-dependent manner. Campylobacter jejuni DC3 inhibition was most evident in CFS harvested at 72 h and produced by co-culture with the pathogen. Neutralisation of the CFS abrogated the observed inhibition. Co-infection with C. jejuni DC3 and L. sakei L14 in vivo, however, failed to inhibit C. jejuni colonisation in chickens.

The results suggest that the anti-C. jejuni effect of L. sakei L14 in chickens may be due to mechanisms other than direct inhibition of growth.

Contagious agalactia (CA) is a disease affecting small ruminants with worldwide distribution and caused by several mycoplasmas, especially M. agalactiae. The main option for systematic diagnosis under monitoring control programmes is the enzyme-linked immunosorbent assay (ELISA) test.

This study was designed to appraise the performance of two commercial indirect ELISA tests using M. agalactiae p48 protein and one using total protein, for antibody detection in small ruminants after natural infection with different M. agalactiae strains. We carried out the test evaluation using sera of confirmed M. agalactiae-positive goats with clinical signs. In addition, test agreement was assessed by kappa between the three commercial ELISA tests.

All three ELISA tests showed high validity scores (Youden’s J: 72.9–84%). The sensitivity values for the P48 protein-based tests were 76.9% and 84.6%, and was 79% for the total protein-based test. The specificity of all tests was 100%. In addition, between the total protein-based ELISA test and the other two ELISA tests based on the P48 protein, the agreement was substantial (kappa: 0.762–0.763) and the agreement between the latter two tests was almost perfect (kappa: 0.93).

The validity parameters for all tests allowed their application for diagnostic purposes in lactating goats excreting M. agalactiae in milk and presenting clinical signs. The agreements show that any of these ELISA tests could be equally well used for diagnosis in programmes against CA.

The aim of the study was to investigate the relationship between biosecurity as scored on the Italian National Animal Welfare Reference Centre (Centro di Referenza Nazionale per il Benessere Animale – CReNBA) checklist and the prevalence of Mycobacterium avium subsp. paratuberculosis, Chlamydophila abortus and Neospora caninum on dairy farms located in Ragusa, Italy.

The checklist was used to assign an animal welfare score to 31 dairy farms. Twenty-one farms with a moderate score (>33%, <66%) formed group 1, and 10 farms with a high score (>66%) were group 2. Blood samples were collected from all cows on each farm to investigate the titres of antibodies against the relevant pathogens. Two-way analysis of variance was applied to assess differences between the two experimental groups and the Mann–Whitney test was applied to evaluate prevalence differences in the tested parasites between the groups.

All tested farms had a score that classified them as either good or excellent. A higher incidence of Neospora caninum was observed in group 1. The incidences of the other two parasites were no different between the two groups.

The CReNBA checklist represents an impartial, reproducible, functional and smart instrument based on risk analysis and assigns a farm a mathematical animal welfare score. Among the parasites tested for, only Neospora caninum had prevalence influenced by biosecurity. Our preliminary results highlighted the positive associations between good animal welfare, high levels of biosecurity, and the prevention of the infectious diseases caused by the parasites in our focus, which are common on dairy farms.

Bovine respiratory disease (BRD) is one of the primary causes of death in young calves. Vaccination against infection by the common bacteria causing BRD is possible; however, the physical condition of the young calves that enables antibody production when stimulated by early immunisation remains to be elucidated.

Healthy young female Holstein calves on a commercial dairy farm were fed a colostrum replacer and administered primary and booster immunisations with an inactivated vaccine against the bacterial pneumonia agents Histophilus somni, Pasteurella multocida and Mannheimia haemolytica. At each immunisation, the body weight and height at the withers were measured and the body mass index (BMI) was calculated. Blood was sampled immediately before immunisation and 3 weeks following the booster. The calves were divided into positive and negative groups based on the antibody titre at the final blood sampling. Maternal antibody titres at the primary immunisation and BMI, nutritional status and oxidative stress at both immunisations were compared between the two groups.

Antibody titre at the primary and BMI at both immunisations were significantly higher in the positive than in the negative group (P < 0.05). Additionally, serum gamma globulin was significantly higher in the positive group (P < 0.05), indicating a strong correlation between maternal antibody and serum gamma globulin levels.

Elevated maternal antibody titre and higher BMI are positive factors for successful early immunisation, for which suitable colostrum may also be fundamental in young calves administered inactivated vaccines.

The prevalence of Dirofilaria repens in dogs in countries bordering Slovenia ranges from 1.5% to 47.3%. The aim of this study was to estimate its prevalence in Slovenian dogs and to present the cases of dirofilariasis diagnosed in humans from 2010 to 2020.

Epidemiological data were collected and blood samples were taken from 465 dogs older than one year and born in Slovenia. A real-time PCR was performed on all samples to detect filarioid DNA, and a D. repens-and D. immitis-specific real-time PCR was performed on positive samples. Blood samples from 446 dogs were tested for Dirofilaria spp. using a modified Knott’s test. Human cases were diagnosed from histological sections of excised subcutaneous nodules. Descriptive statistics were used to characterise the samples. The one-sample nonparametric chi-squared test was used to assess whether categories of a variable were equally distributed.

Three dogs’ samples tested positive for D. repens using the species-specific real-time PCR, while D. immitis DNA was not detected. The modified Knott’s test was positive in two of the three PCR-positive dogs, two of which had never travelled outside Slovenia’s borders. Four human patients with D. repens dirofilariasis were diagnosed. Since their travel history was unknown, autochthonous transmission could not be confirmed.

Our study demonstrated a 0.64% prevalence of D. repens infection in dogs in Slovenia. Two cases could be autochthonous.

Trypanosomosis is an important disease of dromedary camels caused by the pathogenic protozoan Trypanosoma evansi. This study aimed to compare three different tests for its diagnosis in this species: conventional microscopy, the card agglutination test for trypanosomosis/T. evansi (CATT/T. evansi) and real-time PCR.

Whole blood and serum samples collected from 77 dromedary camels of Abu Dhabi, United Arab Emirates, were analysed with the test methods stated. Statistical analysis was done using McNemar’s chi-squared test, and Cohen’s kappa index (κ) was calculated.

We obtained results with positivity of 18% (14/77) by microscopy, 22% by CATT (17/77) and 60% (46/77) by real-time PCR, with the chain reaction detecting at a respectively three- and two-fold greater rate than the other techniques. Analysis of the data revealed a relative sensitivity of 30.4% and 37.0% for microscopy and CATT, respectively, compared to real-time PCR. The difference between the real-time PCR’s sensitivity and those of the other methods was statistically significant, with X² values of 30.03 and 20.1, respectively (df = 1 and P = 0.05 in both cases). Agreement of microscopy results with those of with CATT was good (κ = 0.72; 95% CI = 0.62–0.82). Cohen’s kappa index showed fair agreement of real-time PCR with microscopy (κ = 0.26; 95% CI = 0.16–0.36) whereas it was in poor agreement with CATT (κ = 0.09; 95% CI = 0.02–0.15).

Real-time PCR was found to be more sensitive than microscopy and CATT.

Hypoxia is a common pathological condition after spinal cord injury. Oestrogen-related receptor alpha (ERRα), as a key regulator of energy metabolism and mitochondrial functions, plays an important role in maintaining cell homeostasis. However, its role in hypoxic spinal microglia has not been fully elaborated. This study investigated the receptor’s activity when these cells are hypoxic and used as an in vitro model.

In this study, microglia (BV2) were exposed to cobalt chloride as a hypoxic model, and the inverse agonist of ERRα, XCT790, and pyrido[1,2-α]-pyrimidin-4-one were used to regulate the expression of the receptor to explore the ERRα-related mechanisms involved in hypoxic spinal cord injury (SCI).

ERRα promoted autophagy in BV2 cells and inhibited the activation of the p38 mitogen-activated protein kinase (MAPK) pathway and the expression of anti-inflammatory factors under hypoxic conditions. It also promoted the expression of fibronectin type III domain containing protein 5 (FNDC5).

When a hypoxic SCI occurs, ERRα may maintain the homeostasis of spinal cord nerve cells by regulating autophagy and the p38MAPK/nuclear factor-kappa B cell and FNDC5/brain-derived neurotrophic factor signalling pathways, which are beneficial to the recovery of these cells.