Cultural water and Indigenous water science.

Australia shows the need for more sustainable and just water management.

Cult, herding, and 'pilgrimage' in the Late Neolithic of north-west Arabia: Excavations at a mustatil east of AlUla.

Since the 1970s, monumental stone structures now called mustatil have been documented across Saudi Arabia. However, it was not until 2017 that the first intensive and systematic study of this structure type was undertaken, although this study could not determine the precise function of these features. Recent excavations in AlUla have now determined that these structures fulfilled a ritual purpose, with specifically selected elements of both wild and domestic taxa deposited around a betyl. This paper outlines the results of the University of Western Australia's work at site IDIHA-0008222, a 140 m long mustatil (IDIHA-F-0011081), located 55 km east of AlUla. Work at this site sheds new and important light on the cult, herding and 'pilgrimage' in the Late Neolithic of north-west Arabia, with the site revealing one of the earliest chronometrically dated betyls in the Arabian Peninsula and some of the earliest evidence for domestic cattle in northern Arabia.

Hedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient.

Cellular differentiation is directly determined by concentration gradients of morphogens. As a central model for gradient formation during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in Drosophila wing and eye discs. What is not known is how extracellular Hh spread is achieved and how it translates into precise gradients. Here we show that two separate binding areas located on opposite sides of the Hh molecule can interact directly and simultaneously with two heparan sulfate (HS) chains to temporarily cross-link the chains. Mutated Hh lacking one fully functional binding site still binds HS but shows reduced HS cross-linking. This, in turn, impairs Hhs ability to switch between both chains in vitro and results in striking Hh gradient hypomorphs in vivo. The speed and propensity of direct Hh switching between HS therefore shapes the Hh gradient, revealing a scalable design principle in morphogen-patterned tissues.

Development of trofinetide for the treatment of Rett syndrome: from bench to bedside.

Rett syndrome (RTT) is rare neurodevelopmental disorder caused by mutations in the MECP2 gene that encodes methyl-CpG-binding protein 2 (MeCP2), a DNA-binding protein with roles in epigenetic regulation of gene expression. Functional loss of MeCP2 results in abnormal neuronal maturation and plasticity, characterized by loss of verbal communication and loss of fine and gross motor function, among others. Trofinetide, a synthetic analog of glycine-proline-glutamate, was approved by the US Food and Drug Administration for the treatment of RTT in adult and pediatric patients aged 2 years and older. Here, we present the development of trofinetide from bench research to clinical studies and emphasize how the collaboration between academia, the pharmaceutical industry, and patient advocacy led to the recent approval. The bench-to-bedside development of trofinetide underscores the value of collaboration between these groups in the development and approval of treatments for rare diseases.

2022 AAFP/EveryCat Feline Infectious Peritonitis Diagnosis Guidelines.

CLINICAL IMPORTANCE: Feline infectious peritonitis (FIP) is one of the most important infectious diseases and causes of death in cats; young cats less than 2 years of age are especially vulnerable. FIP is caused by a feline coronavirus (FCoV). It has been estimated that around 0.3% to 1.4% of feline deaths at veterinary institutions are caused by FIP. SCOPE: This document has been developed by a Task Force of experts in feline clinical medicine as the 2022 AAFP/EveryCat Feline Infectious Peritonitis Diagnosis Guidelines to provide veterinarians with essential information to aid their ability to recognize cats presenting with FIP. TESTING AND INTERPRETATION: Nearly every small animal veterinary practitioner will see cases. FIP can be challenging to diagnose owing to the lack of pathognomonic clinical signs or laboratory changes, especially when no effusion is present. A good understanding of each diagnostic test's sensitivity, specificity, predictive value, likelihood ratio and diagnostic accuracy is important when building a case for FIP. Before proceeding with any diagnostic test or commercial laboratory profile, the clinician should be able to answer the questions of 'why this test?' and 'what do the results mean?' Ultimately, the approach to diagnosing FIP must be tailored to the specific presentation of the individual cat. RELEVANCE: Given that the disease is fatal when untreated, the ability to obtain a correct diagnosis is critical. The clinician must consider the individual patient's history, signalment and comprehensive physical examination findings when selecting diagnostic tests and sample types in order to build the index of suspicion 'brick by brick'. Research has demonstrated efficacy of new antivirals in FIP treatment, but these products are not legally available in many countries at this time. The Task Force encourages veterinarians to review the literature and stay informed on clinical trials and new drug approvals.

Differential effects of fluoxetine on the phototactic behavior of 3 amphipod species (Crustacea; Amphipoda).

We document phototactic responses in different amphipod populations of Gammarus minus, Stygobromus tenuis, and Crangonyx shoemakeri, each collected at 2-3 sites within the Washington DC area. We then assessed how baseline phototaxis was altered following either short-term (3-week) or long-term (6-week) exposure to 0.05 µg/L or 0.5 µg/L fluoxetine. Our results classify all species as significantly photonegative, a response that depended solely on the presence, not quality, of light. Short-term fluoxetine exposure caused some animals to become photoneutral, regardless of concentration, while others remained photonegative. Long-term exposure to 0.5 µg/L fluoxetine caused photoneutral behaviors in all surviving populations; exposure to 0.05 µg/L had variable effects. These differential effects were due to a significant effect of population/sampling location on photobehavior. Overall, these results identify species-specific effects of chronic fluoxetine exposure and underscore how the response to light in 7 geographically distinct populations is uniquely tuned to requirements for survival.

Possible Cross-Reactivity of Feline and White-Tailed Deer Antibodies against the SARS-CoV-2 Receptor Binding Domain.

In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2's RBD is antigenically distinct from common human and animal coronaviruses, allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2's RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2's RBD. Surprisingly prepandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were ∼50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing, as cross-reactive antibodies toward SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. IMPORTANCE We report cross-reactive antibodies from prepandemic cats and postpandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2's RBD region. However, we lack conclusive evidence of prepandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

Efficacy and Outcomes of Faecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection in Children with Inflammatory Bowel Disease.

BACKGROUND: Children with inflammatory bowel disease [IBD] are disproportionally affected by recurrent Clostridioides difficile infection [rCDI]. Although faecal microbiota transplantation [FMT] has been used with good efficacy in adults with IBD, little is known about outcomes associated with FMT in paediatric IBD. METHODS: We performed a retrospective review of FMT at 20 paediatric centres in the USA from March 2012 to March 2020. Children with and without IBD were compared with determined differences in the efficacy of FMT for rCDI. In addition, children with IBD with and without a successful outcome were compared with determined predictors of success. Safety data and IBD-specific outcomes were obtained. RESULTS: A total of 396 paediatric patients, including 148 with IBD, were included. Children with IBD were no less likely to have a successful first FMT then the non-IBD affected cohort [76% vs 81%, p = 0.17]. Among children with IBD, patients were more likely to have a successful FMT if they received FMT with fresh stool [p = 0.03], were without diarrhoea prior to FMT [p = 0.03], or had a shorter time from rCDI diagnosis until FMT [p = 0.04]. Children with a failed FMT were more likely to have clinically active IBD post-FMT [p = 0.002] and 19 [13%] patients had an IBD-related hospitalisation in the 3-month follow-up. CONCLUSIONS: Based on the findings from this large US multicentre cohort, the efficacy of FMT for the treatment of rCDI did not differ in children with IBD. Failed FMT among children with IBD was possibly related to the presence of clinically active IBD.

Current Challenges in Fecal Microbiota Transplantation for Clostridioides difficile Infection in Children.

INTRODUCTION: The impact of the 2019 US Food and Drug Administration safety alert involving transmission of multidrug resistant organisms through fecal microbiota transplantation (FMT), and the COVID-19 pandemic on the use of FMT in children, is unknown. METHODS: A survey of pediatric gastroenterologists performing FMT for Clostridioides difficile infection was conducted. RESULTS: Of 36 respondents, 17 (47%) and 30 (83%) changed their FMT practices related to the US Food and Drug Administration safety alert and COVID-19 pandemic, respectively, with 22 (61%) of programs halted. DISCUSSION: The US Food and Drug Administration safety alert and COVID-19 pandemic have substantially influenced the availability and access of FMT for children.

Detection and analysis of tick-borne infections in communal dogs of northwest Zimbabwe.

Domestic dogs (Canis familiaris) may serve as a reservoir or a sentinel for infectious disease pathogens that can affect human and wildlife health. To understand the role of tick-borne diseases in rural and lesser developed regions, we investigated the prevalence of several tick-borne pathogens in communal dogs of Zimbabwe. Blood samples from 225 dogs in northwest Zimbabwe were assessed by serology for Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi, and 241 samples were assessed by polymerase chain reaction (PCR) for Ehrlichia. There was a high seroprevalence (73%) of E. canis-specific antibodies in domestic dogs in northwest Zimbabwe, but follow up analyses via PCR and genetic sequencing indicated only 7.5% of the canines were actively infected with the organism. Whilst indicating that an organism serologically related to E. canis is likely present in the region, this data also shows that the organism is currently present in a relative minority of the domestic dogs in the region. Its presence as evidenced by both serologic and PCR analysis is significant because of the 'one health' paradigm, where humans and wildlife may be affected by the exposure to this pathogen in domestic dogs.

Effect of ultraviolet germicidal irradiation of the air on the incidence of upper respiratory tract infections in kittens in a nursery.

OBJECTIVE: To evaluate the effect of UV germicidal irradiation of the air on the incidence of upper respiratory tract infections (URIs) in kittens in a nursery. ANIMALS: 4- to 8-week-old kittens admitted to a kitten nursery in 2016 and 2018. PROCEDURES: 2 UV germicidal irradiation systems (1 within the heating, ventilation, and air conditioning system and 1 attached to the ceiling) were installed in a kitten nursery. Data were collected on the number of kittens in which a URI was diagnosed by means of a physical examination. The incidence of URIs was compared between 2016, when no UV systems were used, and 2018, when the UV systems were used. RESULTS: The overall incidence of URIs in 2016 was 12.4 cases/100 kitten admissions and in 2018 was 1.6 cases/100 kitten admissions, a significant decrease of 87.1% between the years. CONCLUSIONS AND CLINICAL RELEVANCE: A significant reduction in the incidence of URIs in kittens in a nursery was noted when the UV germicidal irradiation systems were used. Therefore, airborne transmission of feline respiratory pathogens may be more important than has been previously recognized. Ultraviolet germicidal irradiation systems that disinfect the air may be an effective adjunct to standard infection prevention and control protocols in reducing the risk of the transmission of respiratory pathogens among kittens in nurseries and shelters. However, additional studies are needed to confirm the findings reported here.

Betacoronavirus Genomes: How Genomic Information has been Used to Deal with Past Outbreaks and the COVID-19 Pandemic.

In the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to understand the pathogenesis, animal origin and mode of transmission of coronaviruses in the aftermath of the 2002-2003 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-2020 coronavirus disease 2019 (COVID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2. We focus on shared genomic features of the betacoronaviruses and the application of genomic information to phylogenetic analysis, molecular epidemiology and the design of diagnostic systems, potential drugs and vaccine candidates.

Feline Infectious Peritonitis: Update on Pathogenesis, Diagnostics, and Treatment.

Feline infectious peritonitis (FIP) is a mysterious and lethal disease of cats. The causative agent, feline coronavirus (FCoV), is ubiquitous in most feline populations, yet the disease is sporadic in nature. Mutations in the infecting virus combined with an inappropriate immune response to the FCoV contribute to the development of FIP. Diagnosis can be challenging because signs may be vague, clinical pathology parameters are nonspecific, and the gold standard for diagnosis is invasive: histopathology of affected tissue. This article discusses the developments in the understanding of this disease as well as the progress in diagnosis and treatment.

Investigation of the effects of an ultraviolet germicidal irradiation system on concentrations of aerosolized surrogates for common veterinary pathogens.

OBJECTIVE: To determine whether exposure to UV germicidal irradiation (UVGI) reduces concentrations of viable aerosolized microorganisms (attenuated strains of common veterinary pathogens) in a simulated heating, ventilation, and air conditioning (HVAC) system. SAMPLE: 42 air samples seeded with bacteriophage MS2 or attenuated strains of Bordetella bronchiseptica, feline calicivirus, feline herpesvirus-1, canine parvovirus, or canine distemper virus (6/microorganism) or with no microorganisms added (6). PROCEDURES: A simulated HVAC unit was built that included a nebulizer to aerosolize microorganisms suspended in phosphate-buffered water, a fan to produce airflow, 2 UVGI bulb systems, and an impinger for air sampling. Ten-minute trials (3 with UVGI, 3 without UVGI, and 1 negative control) were conducted for each microorganism. Impingers collected microorganisms into phosphate-buffered water for subsequent quantification with culture-based assays. Results for samples yielding no target microorganisms were recorded as the assay's lower limit of detection. Statistical analysis was not performed. RESULTS: The UVGI treatment resulted in subjectively lower concentrations of viable MS2, B bronchiseptica, and canine distemper virus (arithmetic mean ± SD log10 microorganism reduction, 2.57 ± 0.47, ≥ 3.45 ± 0.24, and ≥ 1.50 ± 0.25, respectively) collected from air. Feline herpesvirus-1 was detected in only 1 sample without and no samples with UVGI treatment. Feline calicivirus and canine parvovirus were not detectable in any collected samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results for some surrogates of veterinary pathogens suggested a potential benefit to supplementing manual disinfection practices with UVGI-based air cleaning systems in animal care environments. Further research is needed to investigate the utility of UVGI in operating HVAC systems.

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana.

BACKGROUND: Domestic dogs (Canis familiaris) have the potential to act as disease reservoirs for wildlife and are important sentinels for common circulating pathogens. Therefore, the infectious disease seroprevalence among domestic dogs in northern Botswana may be indicative of pathogen exposure of various wildlife species. The objective of this study was to assess the seroprevalence of Ehrlichia spp., Borrelia burgdorferi, Anaplasma spp., Dirofilaria immitis, canine adenovirus, canine parvovirus, and canine distemper virus in domestic dogs as proxies of disease prevalence in the local wildlife in the Okavango Delta region of Botswana. Statistical analysis assessed crude and factor-specific seroprevalence proportions in relation to age, sex, and geographical location as predictors of seropositivity. Logistic regression was used to identify adjusted predictors of seropositivity for each of the pathogens of interest. RESULTS: Samples from 233 dogs in a total of seven locations in Maun, Botswana, and surrounding villages were collected and serologically analyzed. No dogs were seropositive for B. burgdorferi, while low seroprevalence proportions were observed for Anaplasma spp. (2.2%) and D. immitis (0.9%). Higher seroprevalence proportions were observed for the tick-borne pathogen Ehrlichia spp. (21.0%), and 19.7% were seropositive for canine adenovirus (hepatitis). The highest seroprevalence proportions were for canine parvovirus (70.0%) and canine distemper virus (44.8%). The predictors of seropositivity revealed that adults were more likely to be seropositive for canine adenovirus, canine distemper virus, and canine parvovirus than juveniles, and location was a risk factor for canine adenovirus, canine distemper virus, canine parvovirus, and Ehrlichia spp. CONCLUSIONS: Results indicate that increasing tick control and vaccination campaigns for domestic dogs may improve the health of domestic animals, and potentially wildlife and humans in the Okavango Delta since viral and vector-borne bacterial pathogens can be transmitted between them.

Efficacy of Fecal Microbiota Transplantation for Clostridium difficile Infection in Children.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is commonly used to treat Clostridium difficile infection (CDI). CDI is an increasing cause of diarrheal illness in pediatric patients, but the effects of FMT have not been well studied in children. We performed a multi-center retrospective cohort study of pediatric and young adult patients to evaluate the efficacy, safety, and factors associated with a successful FMT for the treatment of CDI. METHODS: We performed a retrospective study of 372 patients, 11 months to 23 years old, who underwent FMT at 18 pediatric centers, from February 1, 2004, to February 28, 2017; 2-month outcome data were available from 335 patients. Successful FMT was defined as no recurrence of CDI in the 2 months following FMT. We performed stepwise logistic regression to identify factors associated with successful FMT. RESULTS: Of 335 patients who underwent FMT and were followed for 2 months or more, 271 (81%) had a successful outcome following a single FMT and 86.6% had a successful outcome following a first or repeated FMT. Patients who received FMT with fresh donor stool (odds ratio [OR], 2.66; 95% CI, 1.39-5.08), underwent FMT via colonoscopy (OR, 2.41; 95% CI, 1.26-4.61), did not have a feeding tube (OR, 2.08; 95% CI, 1.05-4.11), or had 1 less episode of CDI before FMT (OR, 1.20; 95% CI, 1.04-1.39) had increased odds for successful FMT. Seventeen patients (4.7%) had a severe adverse event during the 3-month follow-up period, including 10 hospitalizations. CONCLUSIONS: Based on the findings from a large multi-center retrospective cohort, FMT is effective and safe for the treatment of CDI in children and young adults. Further studies are required to optimize the timing and method of FMT for pediatric patients-factors associated with success differ from those of adult patients.

Complete Genome Sequence of Bovine Viral Diarrhea Virus-1 Strain Egy/Ismailia/2014, Subtype 1b.

Here, we report the complete genome sequence of bovine viral diarrhea virus-1b (BVDV-1b), strain Egy/Ismailia/2014. The virus genome is composed of 12,217 nucleotides organized as one open reading frame encoding 3,898 amino acids. This report will assist efforts in diagnostics, studying molecular epidemiology, and control of BVDV in Egypt.

Circulation of bovine viral diarrhea virus--1 (BVDV-1) in dairy cattle and buffalo farms in Ismailia Province, Egypt.

INTRODUCTION: Bovine viral diarrhea (BVD) is one of the most economically significant diseases in the bovine industry causing losses due to diarrhea, reproductive disorders, immunosuppression and mortalities. The aim of our investigation was to detect and subtype BVDV from calves on two dairy cattle and two buffalo farms in Ismailia province, Egypt as an indicator of BVDV infection status in the province. METHODOLOGY: A total of 298 blood samples were collected and tested using an optimized one-step, real-time multiplex Taqman-based RT-PCR. All the positive samples by the multiplex real-time RT-PCR were tested using conventional RT-PCR to amplify multiple areas of the genome for further phylogenetic analysis and subtyping. RESULTS: Thirty one (10.4%) of the tested samples were positive for BVDV-1. Only three samples, all from a single dairy cattle farm, had enough viral RNA to be amplified by RT-PCR. The PCR products were sequenced and phylogenetic analysis revealed detection of BVDV-1b. The detected strain is closely related to worldwide BVDV-1b strains, making it difficult to trace its origin. Nucleotide and amino acid alignments of the E2 glycoprotein region of the detected strain with other BVDV-1b strains showed high divergence, with identity ranging from 81.3% to 93.6% and 85.3% to 93.6%, respectively. CONCLUSION: To our knowledge, this is the first report describing the circulation of BVDV-1b in Egyptian dairy cattle populations.

Effect of small interfering RNAs on in vitro replication and gene expression of feline coronavirus.

OBJECTIVE: To evaluate the ability of small interfering RNAs (siRNAs) to inhibit in vitro viral replication and gene expression of feline coronavirus (FCoV). SAMPLE: Cell cultures of Crandell-Rees feline kidney cells. PROCEDURES: 5 synthetic siRNAs that each targeted a different region of the FCoV genome were tested individually and in various combinations for their antiviral effects against 2 strains of FCoV (feline infectious peritonitis virus WSU 79-1146 and feline enteric coronavirus WSU 79-1683) in cell cultures. Tested combinations targeted the FCoV leader and 3' untranslated region, FCoV leader region and nucleocapsid gene, and FCoV leader region, 3' untranslated region, and nucleocapsid gene. For each test condition, assessments included relative quantification of the inhibition of intracellular viral genomic RNA synthesis by means of real-time, reverse-transcription PCR analysis; flow cytometric evaluation of the reduction of viral protein expression in infected cells; and assessment of virus replication inhibition via titration of extracellular virus with a TCID50 infectivity assay. RESULTS: The 5 siRNAs had variable inhibitory effects on FCoV when used singly. Combinations of siRNAs that targeted different regions of the viral genome resulted in more effective viral inhibition than did individual siRNAs that targeted a single gene. The tested siRNA combinations resulted in approximately 95% reduction in viral replication (based on virus titration results), compared with findings in negative control, nontargeting siRNA-treated, FCoV-infected cells. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro replication of FCoV was specifically inhibited by siRNAs that targeted coding and noncoding regions of the viral genome, suggesting a potential therapeutic application of RNA interference in treatment of feline infectious peritonitis.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.