Sequencing methods for HA and NA genes of avian influenza viruses from wild bird feces using Oxford Nanopore sequencing.

We developed a method to determine the sequences of hemagglutinin (HA) and neuraminidase (NA) from RNA extracted directly from wild bird fecal samples, using Nanopore Flongle. We determined the nucleotide sequences and subtypes of HA and NA in 16 and 15 samples respectively, using Flongle. The results of HA and NA subtyping determined by the conventional method were consistent with their subtypes determined by our method, thereby the applicability of this method in the identification of HA and NA subtypes. In addition, the homology between the HA fragments in this and the Sanger methods ranged from 98.5 % to 100 %. Compared with conventional PCR with the Sanger method, this method can easily determine HA and NA subtypes and sequences directly from the fecal samples. It is easier to implement and has lower running costs (USD100$) than other NGS-based methods, making it a useful tool for avian influenza surveillance in wild birds.

The nucleoprotein is responsible for intracerebral pathogenicity of A/duck/Mongolia/47/2001 (H7N1) in chicks.

Avian influenza viruses A/duck/Mongolia/47/2001 (H7N1) (47/01) and A/duck/Mongolia/867/2002 (H7N1) (867/02) were defined as low-pathogenic avian influenza viruses (LPAIVs) using an intravenous pathogenicity test in chickens. On the other hand, the intracerebral pathogenicity indices of 47/01 and 867/02 were 1.30 and 0.00, respectively. A series of reassortant viruses were generated between 47/01 and 867/02, and their intracerebral pathogenicity was compared in one-day-old chicks to identify the protein(s) responsible for the intracerebral pathogenicity of 47/01. The results indicate that the amino acids at positions 50 and 98 of the nucleoprotein are related to the pathogenicity of 47/01 in chicks by intracerebral inoculation. A significant association was found between mortality of the chicks inoculated intracerebrally with 47/01 and virus replication in the lungs and/or brain. These results indicate that the NP of avian influenza viruses may be responsible for intracerebral pathogenicity in the host.

Use of a Participatory Method for Community-Based Brucellosis Control Design in Agro-Pastoral Areas in Tanzania.

Brucellosis is widespread in both humans and livestock in many developing countries. The authors have performed a series of epidemiological studies on brucellosis in agro-pastoral areas in Tanzania since 2015, with the aim of the disease control. Previously, the potential of a community-based brucellosis control initiative, which mainly consisted of the sale of cattle with experience of abortion and vaccinating calves, was assessed as being effective and acceptable based on a quantitative approach. This study was conducted to investigate the feasibility of community-based brucellosis control program using participatory rural appraisals (PRAs) and key-informant interviews. Four PRAs were performed together with livestock farmers and livestock and medical officers in 2017. In the PRAs, qualitative information related to risky behaviors for human infection, human brucellosis symptoms, willingness to sell cattle with experience of abortion, and willingness to pay for calf vaccination were collected, and a holistic approach for a community-based disease control project was planned. All of the communities were willing to implement disease control measures. To avoid human infection, education, especially for children, was proposed to change risky behaviors. The findings of this study showed that community-based disease control measures are promising.

H9N2 influenza virus acquires intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues at the cleavage site of the hemagglutinin and consecutive passages in chickens.

BACKGROUND: Outbreaks of avian influenza (AI) caused by infection with low pathogenic H9N2 viruses have occurred in poultry, resulting in serious economic losses in Asia and the Middle East. It has been difficult to eradicate the H9N2 virus because of its low pathogenicity, frequently causing in apparent infection. It is important for the control of AI to assess whether the H9N2 virus acquires pathogenicity as H5 and H7 viruses. In the present study, we investigated whether a non-pathogenic H9N2 virus, A/chicken/Yokohama/aq-55/2001 (Y55) (H9N2), acquires pathogenicity in chickens when a pair of di-basic amino acid residues is introduced at the cleavage site of its HA molecule. RESULTS: rgY55sub (H9N2), which had four basic amino acid residues at the HA cleavage site, replicated in MDCK cells in the absence of trypsin after six consecutive passages in the air sacs of chicks, and acquired intravenous pathogenicity to chicken after four additional passages. More than 75% of chickens inoculated intravenously with the passaged virus, rgY55sub-P10 (H9N2), died, indicating that it is pathogenic comparable to that of highly pathogenic avian influenza viruses (HPAIVs) defined by World Organization for Animal Health (OIE). The chickens inoculated with the virus via the intranasal route, however, survived without showing any clinical signs. On the other hand, an avirulent H5N1 strain, A/duck/Hokkaido/Vac-1/2004 (Vac1) (H5N1), acquired intranasal pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the HA, followed by two passages by air sac inoculation in chicks. CONCLUSION: The present results demonstrate that an H9N2 virus has the potential to acquire intravenous pathogenicity in chickens although the morbidity via the nasal route of infection is lower than that of H5N1 HPAIV.

Antigenic and genetic analysis of H3N8 influenza viruses isolated from horses in Japan and Mongolia, and imported from Canada and Belgium during 2007-2010.

A/equine/Kanazawa/1/2007 (H3N8), A/equine/Hokkaido/I828/2008 (H3N8) and A/equine/Mongolia/1/2008 (H3N8) were isolated from infected horses. A/equine/Yokohama/aq19/2009 (H3N8) and A/equine/Yokohama/aq13/2010 (H3N8) were isolated from horses imported from Canada and Belgium examined at the Animal Quarantine Service in Yokohama, Japan. In the present study, these five isolates were genetically and antigenically analyzed. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes showed that three isolates from horses in Japan and imported from Canada belonged to the same branch, clade 1 of the Florida sublineage, while the isolates from horses in Mongolia and imported from Belgium belonged to another branch, clade 2 of the Florida sublineage. Reactivity patterns of a panel of monoclonal antibodies to the HA of A/equine/Kanazawa/1/2007 (H3N8) with the five isolates indicate that the HAs of these viruses were antigenically similar to each other and to the reference strains A/equine/La Plata/1/1993 (H3N8) and A/equine/Avesta/1/1993 (H3N8). The present findings indicate that extensive antigenic variation has not accumulated among H3N8 influenza viruses in horses.

Quantitative evaluation of the infection dynamics of bovine brucellosis in Tanzania.

Brucellosis is endemic in Tanzania. A cross-sectional study was conducted at 17 cattle farms in agro-pastoral areas in Tanzania to identify risk factors associated with the within-farm prevalence of bovine brucellosis and to quantitatively assess the infection dynamics through disease modelling. Cattle blood sampling and interviews with farmers using a structured questionnaire were conducted. A total of 673 serum samples were screened using the Rose-Bengal plate test (RBPT), and sero-positivity of RBPT-positive samples was confirmed using a competitive enzyme-linked immunosorbent assay. Zero-inflated binomial regression was performed for univariable and multivariable risk factor analyses of within-farm prevalence. Several susceptible-infectious (SI) models were compared based on deviance information criteria, and age-dependent force of infection (FOI) was measured using age-specific prevalence data for the 10 infection-positive farms. Using the diagnoses of cows on the 17 farms, the basic reproduction number, R0, was also calculated. The farm-level prevalence and animal-level adjusted prevalence were 58.8 % (10/17, 95 % confidence interval: 33.5-80.6 %) and 7.0 % (28/673, 95 % credible interval: 5.7-8.4 %), respectively. The risk factor for high within-farm prevalence was introduction of cattle from other herds. A mathematical model with constant FOI showed the annual probability of infection as 1.4 % (95 % credible interval: 1.0 %-2.0 %). The R0 was 1.07. The constant FOI could have been due to the predominant mode of infection being transmission of Brucella from contaminated aborted materials during grazing. Direct purchase of infected cattle could facilitate efficient transmission between susceptible animals through abortion.

Quantitative Understanding of the Decision-Making Process for Farm Biosecurity Among Japanese Livestock Farmers Using the KAP-Capacity Framework.

In a globalized world, the frequency of transboundary livestock infectious diseases is increasing, and strengthening of farm biosecurity is vital to stabilize food production. The aim of this study was to understand the decision-making process for farm biosecurity among Japanese livestock farmers. Postal surveys using structured questionnaires were conducted on beef, dairy, pig, and layer farms in Hokkaido and Saitama Prefectures, which represent the principal production area and peri-urban Tokyo, respectively, as well as randomly selected broiler farms across Japan. The question items included the attributes of farms and owners, disease experiences, related associations and sources of hygiene information, attitude toward hygiene management, and compliance with the Standards of Rearing Hygiene Management (SRHM). The compliance rates were compared between livestock sectors. Univariable analyses were conducted using combined data from both prefectures, with the compliance rate as the outcome variable and the questionnaire items as explanatory variables, in generalized linear models. Exploratory factor analyses were conducted using the variables with p < 0.2 in the univariable analyses. The factors identified were classified into knowledge, attitude, capacity, practice, and structural equation modeling (SEM) was performed. The questionnaires were completed and returned by 97 and 66 beef cattle, 86 and 136 dairy, 67 and 45 pig, 20 and 39 layer farmers in Hokkaido and Saitama Prefectures, respectively, and 95 broiler farms. The compliance rate was significantly higher among broiler farms (88.9%) compared with the other sectors, followed by pig (77.1%), layer (67.2%), dairy (63.8%), and beef (59.1%) farms in Hokkaido Prefecture, and layer (64.9%), pig (60.0%), dairy (58.5%), and beef (57.6%) farms in Saitama Prefecture. Based on SEM, the decision-making process from greater knowledge to higher attitude, and from higher attitude to greater compliance with the SRHM were significant (p < 0.01) in all sectors. Higher capacity was significantly associated with higher knowledge in dairy, pig,break and layer farms (p < 0.01), and with higher compliance in beef, pig, and layer farms (p < 0.05). These results suggest that the provision of targeted hygiene knowledge to livestock farmers and the support to smallholder farms would improve biosecurity through elevated attitudes and self-efficacy.

Genetic Analyses of an H3N8 Influenza Virus Isolate, Causative Strain of the Outbreak of Equine Influenza at the Kanazawa Racecourse in Japan in 2007.

In August 2007, an outbreak of equine influenza occurred among vaccinated racehorses with Japanese commercial equine influenza vaccine at Kanazawa Racecourse in Ishikawa prefecture in Japan. Apparent symptoms were pyrexia (38.2-41.0 degrees C) and nasal discharge with or without coughing, although approximately half of the infected horses were subclinical. All horses had been shot with a vaccine that contained two inactivated H3N8 influenza virus strains [A/equine/La Plata/93 (La Plata/93) of American lineage and A/equine/Avesta/93 (Avesta/93) of European lineage] and an H7N7 strain (A/equine/Newmarket/1/77). Influenza virus, A/equine/Kanazawa/1/2007 (H3N8) (Kanazawa/07), was isolated from one of the nasal swab samples of diseased horses. Phylogenetic analysis indicated that Kanazawa/07 was classified into the American sublineage Florida. In addition, four amino acid substitutions were found in the antigenic sites B and E in the HA1 subunit protein of Kanazawa/07 in comparison with that of La Plata/93. Hemagglutination-inhibition (HI) test using 16 serum samples from recovering horses revealed that 1.4- to 8-fold difference in titers between Kanazawa/07 and either of the vaccine strains. The present findings suggest that Japanese commercial inactivated vaccine contributed to reducing the morbidity rate and manifestation of the clinical signs of horses infected with Kanazawa/07 that may be antigenically different from the vaccine strains.

Herd-level risk factors associated with Brucella sero-positivity in cattle, and perception and behaviours on the disease control among agro-pastoralists in Tanzania.

Brucellosis is endemic in Tanzania, particularly in agro-pastoral areas. This study investigated the herd-level sero-prevalence and risk factors for Brucella sero-positivity in cattle, and perception and behaviours associated with brucellosis control among agro-pastoralists in Morogoro Region, Tanzania. A cross-sectional study involving herd milk diagnosis by indirect enzyme-linked immunosorbent assay and questionnaire survey was conducted in 124 farms. Questions included potential risk factors, knowledge of brucellosis, willingness-to-pay for cattle vaccination, and item count technique (ICT) for selling behaviour of cows that experienced abortion. Risk factor analysis for Brucella sero-positivity in cattle and analysis of factors associated with willingness-to-pay were conducted using classical tests and generalised linear models. Most farmers had little knowledge about brucellosis (disease name: 13.7%, symptoms: 3.2%, transmission from cattle to human: 2.4%, and Brucella vaccine: 2.4%). The proportion of Brucella sero-positive herd was 44.4% (55/124, 95%CI: 35.5-53.5). No risk factors for Brucella sero-positivity were identified; however, using a veterinary service was identified as a preventive factor (OR = 0.39, 95%CI: 0.18-0.84, p =  0.02). For scenarios of vaccinating all cattle and only calves, 59.7% and 89.5% of farmers were willing to pay for vaccination, respectively. Being a Maasai tribe member was a hesitating factor for vaccinating all cattle (OR = 0.39, 95%CI: 0.19-0.83, p =  0.01) and using a veterinary service was an encouraging factor for vaccinating calves (OR = 4.0, 95%CI: 1.2-13.0, p =  0.02). The ICT found that 45.1% of farmers sold cows that experienced abortion. This estimate was not statistically different from that obtained by direct questioning (34.1%, SE = 7.5%, binomial p value = 0.27, factor score = 1.32), suggesting that farmers did not hesitate to sell such cows. The Maasai conducted more risky behaviours for human infection such as drinking raw milk (p = 0.06) or blood (p <  0.01) and helping delivery with bare hands (p = 0.03) than other tribes. Community-based brucellosis control programmes with calf vaccination may be feasible in the study areas. A One Health approach including the promotion of health education and expansion of veterinary services is crucial for disease control.

Brucellosis Risk in Urban and Agro-pastoral Areas in Tanzania.

Epidemiology of human and animal brucellosis may depend on ecological conditions. A cross-sectional study was conducted to compare prevalence and risk factors of bovine brucellosis, and risky behaviours for the human infection between urban and agro-pastoral areas in Morogoro region, Tanzania. Cattle blood sampling and interviews using a structured questionnaire were conducted with farmers. Rose-Bengal test was conducted for the cattle sera, and positive samples were confirmed with competitive ELISA. Farm-level sero-prevalences were 0.9% (1/106, 95% CI 0.0-5.9%) and 52.9% (9/17, 95% CI 28.5-76.1%) in urban and agro-pastoral areas, respectively. The animal-level-adjusted prevalences were 0.2% (1/667, 95% CI 0.0-1.1%) and 7.0% (28/673, 95% CI 5.7-8.4%) in those areas. The final farm-level model including both areas found two risk factors: history of abortion in the herd (P < 0.01) and cattle grazing (P = 0.07). The animal-level risk factors in agro-pastoral areas were age (P = 0.04) and history of abortion (P = 0.03). No agro-pastoral farmer knew about Brucella vaccine. Agro-pastoralists generally had poorer knowledge on brucellosis and practiced significantly more risky behaviours for human brucellosis such as drinking raw milk (17.6%, P < 0.01) and blood (35.3%, P < 0.01), and helping cattle birth (100%, P = 0.04) than urban farmers (0, 0 and 79.2%, respectively). Intervention programs through education including both human and animal health particularly targeting agro-pastoralists would be needed.