Sero-epidemiology of bluetongue in ruminants raised on private holdings in North-Western Pakistan.

This study investigated the sero-epidemiology of bluetongue in ruminants in North-Western Pakistan. A total of 3,173 serum samples were collected from small (n = 1,651) and large (n = 1,522) ruminants being reared by farmers in 14 districts. Antibodies to bluetongue virus (BTV) were detected using competitive ELISA. The overall prevalence of BTV antibodies was 65%. A significant association (P < 0.05) between the prevalence of BTV antibodies and the risk factors including sex, species, age, area, husbandry practices and breed was shown by univariate analysis. In multivariate analysis, the seroprevalence was 6.5 (95% CL = 3.7-11.4), 5.9 (95% CL = 3.8-9.4) and 2.4 (95% CL = 1.5-3.7) times higher in buffaloes, cattle and goats than sheep, respectively. The seroprevalence was 1.4 (95% CL = 1.1-1.7) times higher in local breeds than in cross/exotic breeds. The seroprevalence was 1.6 (95% CL = 1.1 to 2.3) times higher in sedentary animals than in nomadic animals. The seroprevalence was significantly associated with age. Further work is required to determine the BTV serotypes prevalent in the study area for effective control of the disease.

Prevalence, clinico-epidemiological features, and molecular characterization of bovine papular stomatitis virus in cattle in Iraq.

Bovine papular stomatitis virus (BPSV) is a parapoxvirus that infects cattle, causing skin lesions on the udder and mouth. There have been few studies on the prevalence and molecular characteristics of BPSV in Iraq. Here, we describe the prevalence, phylogenetic analysis, and clinico-epidemiological features of BPSV in cattle in Al-Qadisiyah, Iraq. A total of 264 animals were examined for teat and oral lesions, and BPSV was detected by PCR in 79.9% (211/264) of cattle and calves with skin lesions. The lesions included ulcers, papules, and scabby proliferative areas. The BPSV strains from Iraq clustered phylogenetically with BPSV strains detected in the USA. Further studies are needed to explore the evolution and epidemiology of this virus in the region.

First detection of Tetraparvovirus ungulate 1 in diseased cattle (Chinese Simmental) from Hunan province, China.

Tetraparvovirus is an emerging parvovirus infecting a variety of mammals and humans, and associated with human diseases including severe acute respiratory infection and acute encephalitis syndrome. In the present study, a Tetraparvovirus ungulate 1 (formerly known as bovine hokovirus) strain HNU-CBY-2023 was identified and characterized from diseased Chinese Simmental from Hunan province, China. The nearly complete genome of HNU-CBY-2023 is 5346 nt in size and showed genomic identities of 85-95.5% to the known Tetraparvovirus ungulate 1 strains from GenBank, indicating a rather genetic variation. Phylogenetic and genetic divergence analyses indicated that Tetraparvovirus ungulate 1 could be divided into two genotypes (I and II), and HNU-CBY-2023 was clustered into genotype II. This study, for the first time, identified Tetraparvovirus ungulate 1 from domestic cattle from mainland China, which will be helpful to understand the prevalence and genetic diversity of Tetraparvovirus ungulate 1.

Replication of Akabane virus and related orthobunyaviruses in a fetal-bovine-brain-derived cell line.

Akabane virus (AKAV), Aino virus, Peaton virus, Sathuperi virus, and Shamonda virus are arthropod-borne viruses belonging to the order Elliovirales, family Peribunyaviridae, genus Orthobunyavirus. These viruses cause or may cause congenital malformations in ruminants, including hydranencephaly, poliomyelitis, and arthrogryposis, although their pathogenicity may vary among field cases. AKAV may cause relatively severe congenital lesions such as hydranencephaly in calves. Furthermore, strains of AKAV genogroups I and II exhibit different disease courses. Genogroup I strains predominantly cause postnatal viral encephalomyelitis, while genogroup II strains are primarily detected in cases of congenital malformation. However, the biological properties of AKAV and other orthobunyaviruses are insufficiently investigated in hosts in the field and in vitro. Here, we used an immortalized bovine brain cell line (FBBC-1) to investigate viral replication efficiency, cytopathogenicity, and host innate immune responses. AKAV genogroup II and Shamonda virus replicated to higher titers in FBBC-1 cells compared with the other viruses, and only AKAV caused cytopathic effects. These results may be associated with the severe congenital lesions in the brain caused by AKAV genogroup II. AKAV genogroup II strains replicated to higher titers in FBBC-1 cells than AKAV genogroup I strains, suggesting that genogroup II strains replicated more efficiently in fetal brain cells, accounting for the detection of the latter strains mainly in fetal infection cases. Therefore, FBBC-1 cells may serve as a valuable tool for investigating the virulence and tropism of the orthobunyaviruses for bovine neonatal brain tissues in vitro.

First detection of tick-borne encephalitis virus (TBEV) in raw milk samples in North-Western Iran.

Tick-borne encephalitis virus (TBEV) is a significant cause of flaviviral infections affecting the human central nervous system, primarily transmitted through tick bites and the consumption of unpasteurized milk. This study aimed to assess the prevalence of TBEV and identify new natural foci of TBEV in livestock milk. In this cross-sectional study, unpasteurized milk samples were collected from livestock reared on farms and analysed for the presence and subtyping of TBEV using nested reverse transcription-polymerase chain reaction , alongside the detection of anti-TBEV total IgG antibodies using ELISA. The findings revealed that the highest prevalence of TBEV was observed in goat and sheep milk combined, whereas no TBEV was detected in cow milk samples. All identified strains were of the Siberian subtype. Moreover, the highest prevalence of anti-TBEV antibodies was detected in sheep milk. These results uncover new foci of TBEV in Iran, underscoring the importance of thermal processing (pasteurization) of milk prior to consumption to mitigate the risk of TBEV infection.

Evaluation of bovine coronavirus in Korean native calves challenged through different inoculation routes.

Bovine coronavirus (BCoV) is a pneumoenteric virus that can infect the digestive and respiratory tracts of cattle, resulting in economic losses. Despite its significance, information regarding BCoV pathogenesis is limited. Hence, we investigated clinical signs, patterns of viral shedding, changes in antibody abundance, and cytokine/chemokine production in calves inoculated with BCoV via intranasal and oral. Six clinically healthy Korean native calves (< 30 days old), initially negative for BCoV, were divided into intranasal and oral groups and monitored for 15 days post-infection (dpi). BCoV-infected calves exhibited clinical signs such as nasal discharge and diarrhea, starting at 3 dpi and recovering by 12 dpi, with nasal discharge being the most common symptoms. Viral RNA was detected in nasal and fecal samples from all infected calves. Nasal shedding occurred before fecal shedding regardless of the inoculation route; however, fecal shedding persisted longer. Although the number of partitions was very few, viral RNA was identified in the blood of two calves in the oral group at 7 dpi and 9 dpi using digital RT-PCR analysis. The effectiveness of maternal antibodies in preventing viral replication and shedding appeared limited. Our results showed interleukin (IL)-8 as the most common and highly induced chemokine. During BCoV infection, the levels of IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1ß were significantly affected, suggesting that these emerge as potential and reliable biomarkers for predicting BCoV infection. This study underscores the importance of BCoV as a major pathogen causing diarrhea and respiratory disease.

Detection and Genetic Characterization of Bovine Torovirus in Uruguay.

Bovine torovirus (BToV) is an enteric pathogen that may cause diarrhea in calves and adult cattle, which could result in economic losses due to weight loss and decreased milk production. This study aimed to report the presence, the genetic characterization and the evolution of BToV in calves in Uruguay. BToV was detected in 7.9% (22/278) of fecal samples, being identified in dairy (9.2%, 22/239) but not beef (0.0%, 0/39) calves. BToV was detected in both diarrheic (14%, 6/43) and non-diarrheic (13.2%, 5/38) dairy calves. In addition, BToV was detected in the intestinal contents of 14.9% (7/47) of naturally deceased dairy calves. A complete genome (28,446 nucleotides) was obtained, which was the second outside Asia and the first in Latin America. In addition, partial S gene sequences were obtained to perform evolutionary analyses. Nucleotide and amino acid substitutions within and between outbreaks/farms were observed, alerting the continuous evolution of the virus. Through Bayesian analysis using BEAST, a recent origin (mid-60s) of BToV, possibly in Asia, was estimated, with two introductions into Uruguay from Asia and Europe in 2004 and 2013, respectively. The estimated evolutionary rate was 1.80 × 10-3 substitutions/site/year. Our findings emphasize the importance of continued surveillance and genetic characterization for the effective management and understanding of BToV's global epidemiology and evolution.

First Broad-Range Serological Survey of Crimean-Congo Hemorrhagic Fever among Hungarian Livestock.

(1) Background: Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne disease endemic in Africa, Asia, the Middle East, and the Balkan and Mediterranean regions of Europe. Although no human CCHF cases have been reported, based on vector presence, serological evidence among small vertebrates, and the general human population, Hungary lies within high evidence consensus for potential CCHF introduction and future human infection. Thus, the aim of our pilot serosurvey was to assess CCHF seropositivity among cattle and sheep as indicator animals for virus circulation in the country. (2) Methods: In total, 1905 serum samples taken from free-range cattle and sheep in 2017 were tested for the presence of anti-CCHF virus IgG antibodies using commercial ELISA and commercial and in-house immunofluorescent assays. (3) Results: We found a total of eleven reactive samples (0.58%) from five administrative districts of Hungary comprising 8 cattle and 3 sheep. The most affected regions were the south-central and northwestern parts of the country. (4) Conclusions: Based on these results, more extended surveillance is advised, especially in the affected areas, and there should be greater awareness among clinicians and other high-risk populations of the emerging threat of CCHF in Hungary and Central Europe.

Isolation and Characterization of Contemporary Bovine Coronavirus Strains.

Bovine coronavirus (BCoV) poses a threat to cattle health worldwide, contributing to both respiratory and enteric diseases. However, few contemporary strains have been isolated. In this study, 71 samples (10 nasal and 61 fecal) were collected from one farm in Ohio in 2021 and three farms in Georgia in 2023. They were screened by BCoV-specific real-time reverse transcription-PCR, and 15 BCoV-positive samples were identified. Among them, five BCoV strains from fecal samples were isolated using human rectal tumor-18 (HRT-18) cells. The genomic sequences of five strains were obtained. The phylogenetic analysis illustrated that these new strains clustered with US BCoVs that have been detected since the 1990s. Sequence analyses of the spike proteins of four pairs of BCoVs, with each pair originally collected from the respiratory and enteric sites of one animal, revealed the potential amino acid residue patterns, such as D1180 for all four enteric BCoVs and G1180 for three of four respiratory BCoVs. This project provides new BCoV isolates and sequences and underscores the genetic diversity of BcoVs, the unknown mechanisms of disease types, and the necessity of sustained surveillance and research for BCoVs.

Complete coding region sequence analyses and antigenic characterization of emerging lineage G-IX of foot- and-mouth disease virus serotype Asia1.

Foot-and-mouth disease Virus (FMDV) serotype Asia1 is prevalent in the Indian subcontinent, with only G-III and G-VIII reported in India until 2020. However, in 2019, a novel genetic group within serotype Asia1, designated as G-IX, emerged in Bangladesh, followed by its detection in India in 2020. This report presents analyses of the complete coding region sequences of the G-IX lineage isolates. The length of the open reading frame (ORF) of the two G-IX isolates was 6990 nucleotides without any deletion or insertion. The G-IX isolates showed the highest sequence similarity with an isolate of G-III at the ORF, L, P2, and P3 regions, and with an isolate of G-VIII at the P1 region. Phylogenetic analysis based on the capsid region (P1) supports the hypothesis that G-VIII and G-IX originated from a common ancestor, as speculated earlier. Further, VP1 region-based phylogenetic analyses revealed the re-emergence of G-VIII after a gap of 3 years. One isolate of G-VIII collected during 2023 revealed a codon insertion in the G-H loop of VP1. The vaccine matching studies support the suitability of the currently used Indian vaccine strain IND63/1972 to contain outbreaks due to viruses belonging to G-IX.

Avian Influenza A(H5N1) Virus among Dairy Cattle, Texas, USA.

During March and April 2024, we studied dairy cattle specimens from a single farm in Texas, USA, using multiple molecular, cell culture, and next-generation sequencing pathogen detection techniques. Here, we report evidence that highly pathogenic avian influenza A(H5N1) virus strains of clade 2.3.4.4b were the sole cause of this epizootic.

Sialic Acid Receptor Specificity in Mammary Gland of Dairy Cattle Infected with Highly Pathogenic Avian Influenza A(H5N1) Virus.

In March 2024, the US Department of Agriculture's Animal and Plant Health Inspection Service reported detection of highly pathogenic avian influenza (HPAI) A(H5N1) virus in dairy cattle in the United States for the first time. One factor that determines susceptibility to HPAI H5N1 infection is the presence of specific virus receptors on host cells; however, little is known about the distribution of the sialic acid (SA) receptors in dairy cattle, particularly in mammary glands. We compared the distribution of SA receptors in the respiratory tract and mammary gland of dairy cattle naturally infected with HPAI H5N1. The respiratory and mammary glands of HPAI H5N1-infected dairy cattle are rich in SA, particularly avian influenza virus-specific SA α2,3-gal. Mammary gland tissues co-stained with sialic acids and influenza A virus nucleoprotein showed predominant co-localization with the virus and SA α2,3-gal. HPAI H5N1 exhibited epitheliotropism within the mammary gland, and we observed rare immunolabeling within macrophages.

Effect of doubled dose administration of foot-and-mouth disease vaccine against heterologous virus infection in cattle.

Vaccination is a feasible approach for controlling foot-and-mouth disease (FMD). In FMD-free countries, vaccines are stored as a precautionary measure to control potential outbreaks. However, the challenge lies in pre-stocking optimal vaccines against the newly emerging strains. This study examined the potency of pre-stocked vaccines administered at elevated doses during emergencies. We vaccinated the cows with either a single or double trivalent vaccine dose containing two serotype O and one serotype A strains. Subsequently, vaccinated and unvaccinated cows were exposed to virulent strains of serotype O (O/JPN/2010; topotype Southeast Asia/Mya-98 lineage) or A (A/IRN/2016; topotype ASIA/G-VII lineage), which were genetically and antigenically distinct from the vaccine strains. Following challenge infections, all cows that received a single dose vaccination exhibited vesicular lesions with excreted viruses in the oral and nasal discharges. However, a substantial reduction was observed in the total clinical scores and virus titers in the sera and nasal discharges compared to those in the unvaccinated group. Cows receiving a doubled dose vaccination were completely protected from infection with O/JPN/2010 or demonstrated a significant decrease in viral shedding and clinical scores against A/IRN/2016. To note, vesicular lesions harbor significant amounts of viruses; thus, by mitigating their formation, viral transmission can be impeded, thereby slowing viral spread in the field. Furthermore, increasing the vaccine dose induced higher neutralizing antibody titers against heterologous strains. These findings suggest an alternative strategy for the effective management of future epidemics using pre-stocked vaccines.

Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

Seroepidemiology of bovine alphaherpesvirus 1 (BoAHV-1) in commercial and smallholder dairy herds in north Shewa, central highlands of Ethiopia.

Bovine alphaherpesvirus 1 (BoAHV-1) is the most important respiratory and reproductive disease-causing pathogen in dairy cattle. Despite BoAHV-1 has become widespread and a major challenge to the dairy industry, little is known about its epidemiology in dairy herds in Ethiopia. A cross-sectional study was conducted from November 2022 to May 2023 to determine the seroprevalence and potential risk factors associated with BoAHV-1 seropositivity in dairy herds in North Shewa, the central highlands of Ethiopia. A total of 511 blood samples were collected from randomly selected cattle herds (n = 142) and examined antibodies against BoAHV-1 using ELISA test. A retrospective survey was also done to gather information related to reproductive disorders. The overall seroprevalence of BoAHV-1 was 61.84% (95% CI: 57.53-65.97) at the animal level and 85.21% (95% CI: 78.28-90.21) at the herd level. Multivariable logistic analysis revealed that the risk of being BoAHV-1 seropositive was nine times higher in cows older than six years (OR = 9.16; 95% CI: 3.09-27.16; P = 0.000), five times higher (OR = 4.51; 95% CI: 1.23-16.53; P = 0.019) in cows with a history of abortion, three times higher (OR = 2.75; 95% CI: 1.72-4.22; P = 0.029) in cows with a history of retained fetal membrane, and three times higher (OR = 2.83; 1.86-9.31; P = 0.03) in animals with clinical signs of ocular and/or nasal discharge. This study demonstrates a significant circulating of BoAHV-1 in the dairy cattle population in study districts. Thus, a comprehensive approach that includes strict farm biosecurity and vaccination should be practiced for effective BoAHV-1 control and prevention and to promote the growing dairy industry in the central highlands of Ethiopia.

Leptospirosis as a cause of infertility in Uruguayan beef cattle.

Leptospirosis, caused by pathogenic spirochetes of the genus Leptospira spp., is a globally significant zoonotic disease that affects humans and animals. In cattle, leptospirosis is associated not only with overt clinical manifestations but also with reproductive diseases, including infertility. This study assesses the potential correlation between leptospirosis and infertility in Uruguayan beef cattle. A case-control study involved 31 beef herds with no prior history of Leptospira vaccination. In each herd, veterinarians identified 10 non-pregnant (cases) and 25 pregnant cows (controls) using ultrasound, and blood and urine samples were collected from each cow. Serological diagnosis was performed using the Microscopic Agglutination Test (MAT), and quantitative PCR (qPCR) was used to assess Leptospira excretion. Additionally, antibodies against bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis (IBR) were tested. The results demonstrated an association between seropositivity to the Sejroe serogroup (cut-off 1:200) and infertility in cattle (OR=1.31; p-value=0.06). Furthermore, the level of Leptospira excretion (qPCR) in urine was associated with increased infertility risk, with cows excreting over 100 copies per mL of urine having the highest odds of infertility (OR=2.34; p-value<0.01). This study suggests a potential association between leptospirosis and infertility in Uruguayan beef cattle, emphasizing the importance of both serological and molecular diagnostics for assessing reproductive health in cattle herds. Future research should explore the impact of Leptospira serogroups on other reproductive disorders in cattle.

Parapoxvirus species revisited by whole genome sequencing: A retrospective analysis of bovine virus isolates.

Parapoxviruses (PPV) of animals are spread worldwide. While the Orf virus (ORFV) species is a molecularly well-characterized prototype pathogen of small ruminants, the genomes of virus species affecting large ruminants, namely Bovine papular stomatitis virus (BPSV) and Pseudocowpox virus (PCPV), are less well known. Using Nanopore sequencing we retrospectively show the whole genome sequences (WGS) of six BPSV, three PCPV isolates and an attenuated ORFV strain, originating from different geographic locations. A phylogenetic tree shows that the de novo assembled genomes belong to PPV species including WGS of reference PPV. Remarkably, Nanopore sequencing allowed the molecular resolution of inverted terminal repeats (ITR) and the hairpin loop within the de novo assembled WGS. Additionally, peculiarities regarding map location of two genes and the heterogeneity of a genomic region were noted. Details for the molecular variability of an interferon response modulatory gene (ORF116) and the PCPV specificity of gene 073.5 are reported. In summary, WGS gained by Nanopore sequencing allowed analysis of complete PPV genomes and confident virus species attribution within a phylogenetic tree avoiding uncertainty of limited gene-based diagnostics. Nanopore-based WGS provides robust comparison of PPV genomes and reliable identity determination of new Poxviruses.

Quantification of ovine gammaherpesvirus 2 in clinical cases of cattle with sheep-associated malignant catarrhal fever.

Ovine gammaherpesvirus 2 (OvGHV2) produces sheep-associated malignant catarrhal fever (SA-MCF), a frequently lethal, lymphoproliferative disease that is characterized by widespread vascular lesions. Most studies that evaluated the viral load in tissues of animals with SA-MCF were done in the Northern Hemisphere, with scant information from the Southern part of the globe. This study investigated the viral load of OvGHV2 in the tissues of cattle and an underdeveloped fetus with SA-MCF from three distinct biomes of Brazil. All animals had clinical and histopathological manifestations consistent with SA-MCF. Molecular testing identified the OvGHV2 tegument protein and glycoprotein B genes in the tissues of all animals and the fetus. Viral quantification based on the DNA polymerase gene detected elevated loads of OvGHV2 in tissues with histopathological evidence of SA-MCF and organs with unknown histological data, except for the tissues of the fetus, where the viral load was comparatively reduced. The viral loads detected in multiple organs of cattle from this study with SA-MCF are consistent with those identified in different animal species from the USA and Europe. The detection of a low viral load of OvGHV2 in fetal tissue confirmed transplacental dissemination since elevated viral loads were detected in multiple tissues of the cow with SA-MCF. Furthermore, the elevated viral loads detected in the pulmonary tissues of cattle with interstitial pneumonia indicate that OvGHV2 is an inductor of pulmonary disease in cattle.