Veterinaria Italiana <p>A quarterly peer-reviewed journal devoted to veterinary public health and other aspects of veterinary science and medicine, Veterinaria Italiana is published by the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (Istituto G. Caporale) in Teramo, Italy.</p> Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale en-US Veterinaria Italiana 0505-401X Larvae and adult flies of Rhinoestrus purpureus and R. usbekistanicus: morphology and pupation (Diptera: Oestridae) <p><em>Rhinoestrus</em> species larvae are considered obligatory parasites of the nasal cavities of equine. This type of myiasis is characterised by sneezing, coughing, olfactory nerve damage and encephalomyelitis. Also, it has a zoonotic importance as the larvae might cause ophthalmomyiasis and conjunctivitis in human. While few studies describing <em>R. purpureus</em> adult fly antennal sensillae are available, the <em>R. usbekistanicus</em> antennal sensillae have never been described. Also, scanty data are available on the adult flies of <em>Rhinoestrus</em> species morphology. For this reason, the current study aimed at identiying and comparing different <em>Rhinoestrus</em> species (larvae, adult flies and time of pupation). Using light and scanning electron microscopy, we have evidenced differences between <em>R. purpureus</em>, <em>R. usbekistanicus</em> larvae in spination pattern, shape of spines, peritremes shape and ultrastrucures. The study also showed that for both species the pupa required 15‑21 days at room temperature to develop into adult flies, identified the adult male flies and female external genitalia have been identified. As well, the gross features of <em>R. purpureus</em> and <em>R. usbekistanicus</em> adult flies which included the disposition of the parafrontalia and parafascialia tubercles, mesonotal weals, wings and abdominal pellonisityhave been characterised and the sensillae compared.</p> Mohammed I. Abdel Rahman Copyright (c) 2022 Veterinaria Italiana 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2085.12058.2 Study of fear and fear evoking stimuli in a population of domestic dogs in Iran: A questionnaire-based study <p>General and social acceptance, condition, and rules for dog ownership in Iran are different from western countries. So, this phenomenon leads dissimilar fear and fear evoking stimuli in dogs. So, stress and fear‑related factors and dog’s behavioral problems is not fully studied in Iran. Thus, the aim of the current study was to measuring fear‑related factors in dogs in Iran. In the first part of this study demographic information of the owners and dogs were collected using questionnaire from clinics and veterinary hospitals in Tehran. In the second part behavioral tests was done. Questionnaire data were recorded based on 4 items as neuroticism, fear caused by other dogs, fear caused by human and separation behavior. The social contact test was used to determine dog’s experience for greeting, cooperation, and handling. According to the results, 69.3% of the dog owners were women and 30.7% men. 84.1% of the dogs were small breeds and 15.9% large‑breed dogs. 63.6% of the owner’s complained for impact of laws and prohibitions on their dogs' access to outdoor. The score for greeting, cooperation and handling were 1.76 ± ± 0.93, 3.06 ± 1.25 and 2.44 ± 1.21, respectively. Mean score for neuroticism, fear caused by other dogs, fear caused by human and separation behavior were 29.69 ± 9.24, 14.75 ± 5.38, 9.84 ± 4.05 and 6.57 ± 3.05, respectively. Principal component analysis (PCA) revealed fear using neuroticism, fear caused by other dogs and human were 1st fear related factor while separation‑related behavior was the 2<sup>nd</sup> priority (P = 0.001). Small‑breed dogs had more separation behavior and fear caused by other dogs compared to the large‑breed dogs (P = 0.001). These findings suggested keeping the dogs indoor with limit access to a yard had negative impact on dog’s behaviors. Legal restrictions have adverse effect on fear behavior in dogs. Determination of fear‑related factors might prove useful for dog’s behavioral intervention in Iran.</p> Mohammad Amir Qiasvand Samad Alimohammadi Shahin Hassanpour Copyright (c) 2022 Shahin Hassanpour 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2567.15853.2 Multidrug resistant enterohaemorrhagic Escherichia coli serogroups in the faeces of hunted Wildlife, Abeokuta, Nigeria <p>Wildlife plays significant roles in the dissemination and zoonotic transmission of pathogens. The enterohaemorrhagic Escherichia coli (EHEC) are associated with complicated cases of food‑borne illnesses. This study investigated the presence of EHEC serogroups (O26, O45, O103, O145, O91, O111, O128, O121 and O157) in wildlife species: cane rats (Thryonomys swinderianus), royal antelope (Neotragus pygmaeus), African giant rats (Cricetomys gambianus) and waterbuck (Kobus ellipsiprymnus). EHEC and non‑EHEC isolates from these wildlife sources were tested for susceptibility to antimicrobial agents. Overall, 127 (83.0 %) out of 153 samples yielded E. coli. Nine (5.9%) samples were positive for EHEC belonging to three serogroups as follows: O26 (n = 2), O111 (n = 2) and O103 (n = 5). The EHEC isolates were from cane rats (n = 6) and royal antelope (n = 3) and possessed virulence‑associated genes stx1 (77.8%), stx2 (100.0%), eaeA (100.0%) and hlyA (100.0%). Overall, 127 E. coli isolates showed resistance to ampicillin (99.2%), ceftiofur (90.6%), tetracycline (90.0%), cephalexin (87.4%), cefotaxime (50.4%), streptomycin 42.5%, ceftazidime (41.7%), nalidixic acid (37.0%), ciprofloxacin (43.6%), amoxicillin/clavulanic acid (32.3%), gentamicin (27.6%), sulphamethoxazole/trimethoprim (25.2%), norfloxacin (17.3%) and chloramphenicol (11.0%). The role of wildlife in the dissemination and transmission of antimicrobial resistant and zoonotic bacteria should not be neglected for effective preventive and control strategies.</p> Olufemi Ernest Ojo Elizabeth Adesola Amosun Oluwadaisi Oluwaseyi Opebiyi Mufutau Atanda Oyekunle Morenike Atinuke Dipeolu Ebenezer Babatunde Otesile Copyright (c) 2022 Veterinaria Italiana 2022-12-30 2022-12-30 58 2 10.12834/VetIt.1990.12087.2 Estimation of the risk of fipronil ingestion through the consumption of contaminated table eggs for the Italian consumer <p>Fipronil is an insecticide which is not approved for use in any food‑producing animal species in the European Union (EU). However, the inappropriate use of fipronil in mites’ disinfestation products utilized in poultry farms in the Netherlands and other EU countries in 2017, led to the detection of residues of this pesticide in eggs across Europe. In Italy, a national monitoring plan was established to verify the possible misuse of fipronil in Italian laying hens. Out of 577 sampled farms, 23 eggs resulted contaminated (4.0%; 95% CI: 2.7%‑5.9%). A higher prevalence of contamination was observed in flocks kept on cage (8.7%; 95% CI: 6.0% ‑ 12.4%) than on ground (1.6%; 95% CI: 0.7% ‑3.7%); Chi‑square = 16.1; P &lt; 0.001). The results allowed developing a stochastic model for estimating the risk of fipronil ingestion through the consumption of contaminated table eggs for the Italian consumer. The probability that an individual ingests a dose of fipronil greater than the acute reference dose (ARfD, equal to 0.009 mg/kg body weight) was assessed as very low, ranging from values very close to 0 in people with more than 10 years of age and 0.0007 in infants less than 3 years.</p> Simona Iannetti Rossana Scarpone Francesca Dall'Acqua Roberta Rosato Francesco Chiumiento Daniela Cioci Giacomo Migliorati Daniela Morelli Paolo Calistri Copyright (c) 2022 Simona Iannetti 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2416.16804.2 Molecular characterization of Ornithobacterium rhinotracheale strains isolated from chickens in the Northwestern Iran <p><em><span id="page1287R_mcid20" class="markedContent"></span>Ornithobacterium rhinotracheale</em> is the etiological agent of chickens and turkeys’ respiratory diseases, reduction of eggs, growth retardation, and death. Present research aimed to conduct the isolation, recognition, and molecular investigations of the bacterium in commercial broiler chicken flocks in East Azerbaijan province, Northwest of Iran, by the partial sequencing of 16S rRNA gene, ERIC‑PCR, and RAPD‑PCR with the OPG11 and M13 primers. We obtained 330 specimens from tracheal swabs of 33 slaughtered broiler flocks, of which we found 14 isolates (4.24%) of five flocks (15.15%) to be <em>O. rhinotracheale</em>. Typing by RAPD assay with the OPG11 primer, and ERIC‑PCR, classified the isolates in two types of 1 and 2 molecular patterns, most of which belonged to type 1. However, the M13 primer‑based RAPD technique was inappropriate for distinguishing and categorizing the isolates and generating all of them in the same pattern. In a phylogenetic analysis of <em>O. rhinotracheale</em> based on 16S rRNA sequences, the strains generated three clusters (I‑III), in which all of the studied isolates fell in one cluster (cluster I). Based on the results obtained from the RAPD and ERIC‑PCR assays, the genetic patterns of broiler‑chicken‑isolated <em>O. rhinotracheale</em> strains in Northwestern Iran had no significant differences.</p> Mkh Mohammad Karim Copyright (c) 2022 mkh mohammad karim 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2504.15303.1 Transition of dominant canine parvovirus genotype from 2b to 2c in Vietnamese dogs <p><span id="page1980R_mcid16" class="markedContent"></span>Canine parvovirus (CPV) is one of the most important pathogens causing enteritis in dogs. Although there have been a few reports of CPV in Vietnam, recent information on CPV infection in domestic dogs in Vietnam is limited. Faecal samples collected from 30 diarrheic and 50 healthy dogs were examined by PCR for detection of CPV DNA. The prevalence of CPV in diarrheic dogs (43.3%, 13/30) was significantly higher than in healthy dogs (4.0%, 2/50), indicating that CPV was a major cause of diarrhoea in domestic dogs. Genotyping of 15 CPV strains showed that both CPV‑2a and CPV‑2c were circulating and that CPV‑2c was a dominant CPV variant in Vietnam. Virus isolation was performed from faecal samples using A72/cSLAM cells, and nine CPV strains were successfully isolated. The dominant genotype spreading among Vietnamese dogs has changed from CPV‑2b to CPV‑2c.</p> Dung Nguyen Van Thanh Dinh Ha Le Ken Maeda Copyright (c) 2022 Dung Nguyen Van 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2237.13437.2 Salmonella enterica diversity and antimicrobial resistance profile in broiler slaughterhouse by-products <p>The aim of this study was to investigate the presence of <em>Salmonella enterica</em> in by‑products (feathers, spleen, cecum, and crop) from broiler slaughterhouses as well as to determine the antimicrobial resistance profile of the identified serovars. Forty‑four lots of broilers in nine slaughterhouses located in the central‑west region of Brazil were evaluated. Samples of spleen, feathers, cecum, and crop were collected in a pool and a total of 1,232 samples were evalueted. These were processed for conventional bacterial isolation and subjected to biochemical and serological tests to identify serovars. The identified serovars were subjected to the antimicrobial susceptibility test, where nine different antimycotics were investigated. <em>Salmonella enterica</em> was identified in 7.1% (87/1,232) of all evaluated samples, mostly in feathers (12.3%) and spleen (8.1%). The most frequent serovars were Schwarzengrund (29.9%), Agona (25.4%), Mbandaka (12.7%) and Anatum (8.1%). Nine serovars showed resistance to at least one antimicrobial, especially serovars Mbandaka, Infantis and Typhimurium. Amoxicillin and tetracycline were not effective in inhibiting at least five and four serovars, respectively.</p> Juliana Bonifácio Alcântara Poliana Carneiro Martins Eduardo de Paula Nascente Marcos B. Café Lívia Mendonça Pascoal Amanda Vargas Teles Valéria de Sá Jayme Maria Auxiliadora Andrade Copyright (c) 2022 Juliana Bonifácio Alcântara, Poliana Carneiro Martins, Eduardo de Paula Nascente, Marcos B. Café, Lívia Mendonça Pascoal, Amanda Vargas Teles, Valéria de Sá Jayme, Maria Auxiliadora Andrade 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2434.14968.1 Assessment of Paratuberculosis international official reporting in Europe using the information supplied to the WOAH by National Veterinary Services <p>The present study characterizes the epidemiological situation of Paratuberculosis (PTB) in Europe during the last 24 years, using the information officially reported to the World Organisation for Animal Health (WOAH) by veterinary services of the European countries. The prevalence of PTB at country level was described during the study period. A Cox proportional hazards (PH) regression analysis was implemented to evaluate the notification behaviours. Results from this work indicate that the most affected countries are in Southern and Western Europe, whereas PTB presence appears lower in Northern and Eastern Europe. PTB was routinely declared as a notifiable disease in 65% of the countries. Less than 50% of the countries routinely implemented passive surveillance, and only 19%, reported active surveillance for disease detection. Results from the Cox PH regression indicate that the Gross National Income (GNI) per capita and the application of active surveillance significantly influence the recurrence of PTB reporting. In countries with lower and upper middle income, the hazard of recurrence is 0.13 and 0.18 times lower than in countries with high income. The hazard of recurrence in countries that infrequently and moderately applied active surveillance is 1.99 and 1.65 times higher than in countries that routinely applied active surveillance. Findings from this work highlight an important variation in reporting behaviours, disease status and surveillance across Europe.</p> Angela Fanelli Michela Galgano Alessio Sposato Domenico Buonavoglia Copyright (c) 2022 Angela Fanelli, Michela Galgano, Alessio Sposato, Domenico Buonavoglia 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2625.16709.3 Allelic polymorphisms of the BoLA-DRB3 gene and resistance to brucellosis in Kazakh cattle <p>The Authors investigated polymorphism of the bovine BoLA‑DRB3 gene in connection with resistance or susceptibility to brucellosis of two Kazakh meat breeds, Auliekol and Kazakh Whiteheaded breeds, using PCR‑RFLP. In Auliekol cattle (n = 158), 22 alleles were detected in the brucellosis group, and 24 alleles were shown in the healthy group. BoLA-DRB3 alleles *3, *4, *19, *21 were more common in healthy animals, while <em>Brucella</em>‑positive cattle were more frequently carriers of alleles *7, *10, *18. In Kazakh Whiteheaded cattle (n = 146), 21 alleles were detected in infected and 23 alleles in healthy cattle. Alleles *3, *8, *21 significantly predominate in healthy cattle, while alleles *7, *11, *16 are typical for animals with brucellosis. This study identified BoLA-DRB3 alleles associated with genetic resistance (*3 and *21) and susceptibility (*7) to brucellosis; remarkably, resistance alleles are shared by two important meat breeds of Kazakhstan.</p> Akmaral Adambayeva Akhmetzhan A. Sultanov Irina Ya. Nam Vladimir V. Zayakin Copyright (c) 2022 Akmaral Adambayeva, Akhmetzhan A. Sultanov, Irina Ya. Nam, Vladimir V. Zayakin 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2528.15313.1 First report of paratuberculosis (Johne’s disease) in livestock farms of river buffaloes (Bubalus Bubalis) in Nineveh, Iraq <p><span id="page995R_mcid44" class="markedContent"></span>The present study was designed to investigate <em>Mycobacterium avium</em> subsp. <em>paratuberculosis</em> (MAP) in dairy buffalo herds from six different geographical areas in Nineveh, Iraq. A total of 87 individual faecal samples from river buffaloes, representing 12 dairy herds, were investigated for detection of MAP using cultural, Ziehl‑Neelsen and MAP‑specific PCR‑based methods. Overall, MAP was detected at a higher frequency at herd‑level (4/12; 33%) compared to the total individual faecal samples (14/87; 16%) with a cell density ranging from 10<sup>1</sup> to 10<sup>3</sup> CFU g<sup>‑1</sup>. A significantly (p &lt; 0.05) higher frequency (9/17; 53%) of MAP was observed in faecal samples collected from clinically diseased as compared to healthy (5/70; 7%) buffaloes selected for the study. However, no statistically significant difference (p ≥ 0.05) was observed in the frequency of MAP occurrence between clinical (9; 64%) and apparently healthy (5; 36%) cases. This report, which is the first MAP study based on data from Iraqi dairy buffalo herds suggests that MAP transmission is a significant health risk for grazing livestock. In conclusion, this study would help farm owners and regulatory authorities to realise the importance of developing and applying best farm management practices in order to prevent transmission of MAP to healthy animals and the environment. In addition, effective diagnostic tests should be taken into account when carrying out the screening tests.</p> <p> </p> Abdulwahed Ahmed Hassan Mohammed Rahawy Layth Mahmoud Alkattan Izhar U.H. Khan Amir Abdulmawjood Michael Bülte Copyright (c) 2022 Veterinaria Italiana 2022-12-30 2022-12-30 58 2 10.12834/VetIt.1866.9913.1 Control measures to reduce Neospora caninum abortions in dairy farms: a pilot study in Iran <p><em>Neospora caninum</em> plays an important role in abortion and economic losses in dairy industry worldwide. The main target of this work was to detect the infection rate of <em>N. caninum</em> in various hosts in dairy farms for identifying the risk factors and applying appropriate control programs to reduce the number of abortions. The study was conducted in dairy farms with high incidence of abortion in Hamedan province, West of Iran. After the primary assessment, we conducted a controlling program for reducing the <em>Neospora</em>‑infection rate and associated abortions. Before implementing the control program, the seropositivity was 24.8% in cows (N=476 distributed in 10 farms) and 8.6% (N=185) in dogs. Abortion occurred in 3.57% of pregnant cows. 94.1% of aborted cows were positive for <em>Neospora</em>‑infection. Based on molecular technique, the infection rate was detected in 7.3% of cats (N=41), in 25% of pigeons (N=19)and in 11.8% of rodents (N=51). After the implementation of neosporosis control programs in the farms, the seropositivity of <em>N. caninum</em> decreased to 8.2% in cows and 2.9% in dogs. After the one‑year follow‑up, no cases of abortions were reported in the farms. This was the first parallel evaluation of <em>Neospora</em>‑infection and controlling programs in Iranian dairy farms. Regular control of rodents, retesting of seronegative animals and farm biosecurity measures are recommended for reducing the abortion incidence. The access of dogs to the herd and to aborted materials should be restricted. <br style="font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px;" /><br /></p> Jamal Gharekhani Mohammad Yakhchali Copyright (c) 2022 Jamal Gharekhani, Mohammad Yakhchali 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2327.14464.2 Prevalence of urinary shedders and characterization of pathogenic Leptospira among cattle population in Tamil Nadu ‑ Implications for control <p><span id="page1109R_mcid19" class="markedContent"></span>Bovine leptospirosis causes jaundice, mastitis, infertility, abortion, and death of infected animals. This research aimed to study the status of urinary shedders of pathogenic <em>Leptospira</em> among the cattle population and identify the infecting serogroup circulating in the state of Tamil Nadu (India). A total of 305 blood and 305 urine samples were collected from organized farms (n = 44), individually housed animals (n = 81) and animals from the slaughterhouse (n = 180). Microscopic agglutination test was carried out to detect anti‑leptospiral antibodies. Dark‑field microscopic examination and culture of urine were done to detect and isolate the <em>Leptospira</em>. The isolated <em>Leptospira</em> were identified by cross‑agglutination test and gene sequencing. PCR and real‑time PCR were carried out to detect leptospiral genomic DNA in urine samples to detect the shedders. The anti‑leptospiral antibodies were detected in 6.2% of animals. The <em>Leptospira</em> genomic DNA was detected in 9.2% (28/305) of urine samples. Of the 28 <em>Leptospira</em> positive urine samples, 39.2% were from animals with clinical signs suggestive of leptospirosis and 60.8% <em>Leptospira</em> positive samples were from slaughterhouse animals. The <em>Leptospira</em> isolated were identified as Leptospira interrogans serogroup Sejroe and Hebdomadis. The present study demonstrates the need to include leptospirosis in cattle health surveillance programmes to prevent leptospirosis and renal carriage by vaccination.</p> K. Senthilkumar G. Ravikumar Aravindh Babu R. Parthiban Copyright (c) 2022 K.SENTHILKUMAR 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2478.16135.1 A comprehensive meta-analysis of Brucella infections in aquatic mammals <p><span id="page2007R_mcid35" class="markedContent"></span>The presence of <em>Brucella</em> infections was documented in a large number of aquatic mammals, affecting wild animals living in oceans, seas, lakes and rivers within both northern and southern hemispheres. Through meta‑regression analysis, this study provides a comprehensive view of the prevalence of <em>Brucella</em> spp. in aquatic mammals, identifying risk subgroups as well as most common sampling and testing methods. <em>Brucella ceti</em> and <em>Brucella pinnipedialis</em> represent the main marine <em>Brucella</em> spp., with documented enzootic potential, for which information on standardized diagnostic methods for the implementation of efficient screening and monitoring programs is needed. A total of 71 articles investigating the occurrence of brucellosis in aquatic mammals since 1987, have met the inclusion criteria and have been included in this study. The prevalence of brucellosis in males (30.42%) was significantly higher than females (18.59%). The family of Delphinidae was the most studied among aquatic mammals with a total prevalence of 39.66%. Our meta‑regression analysis showed a strong and significant association between the prevalence of Brucella spp. in mammals and water temperature (C = 0.02, p value = 0.003), while no significant correlation was found with water salinity (C = ‑ 0.09; p value = 0.10). At least 130 species of aquatic mammals have been identified as potential hosts for <em>Brucella</em> spp. There is no systematic veterinary inspection and global or local requirements for the monitoring of brucellosis in aquatic mammals. The association of brucellosis prevalence and water temperature warrants further studies to assess the potential direct and indirect impacts of climate change on brucellosis in aquatic mammals. This study would help to determine the basis of adaptive management strategies in order to control enzootic brucellosis in wild aquatic mammals.</p> Maryam Dadar Youcef Shahali Yadolah Fakhri Jacques Godfroid Copyright (c) 2022 Jacques Godfroid, Maryam Dadar, Youcef Shahali, Yadolah Fakhri 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2427.14954.2 TF-Test techniques for the laboratory diagnosis of gastrointestinal parasites of humans and animals <p>Intestinal parasites inhabit the intestinal tract of humans and animals, causing damages whose severity depends on several factors related to the parasite and the host. Immunocompromised individuals are more likely to develop severe forms of parasitic infestation. The diagnosis of the gastrointestinal parasitosis is mainly performed by the examination of the feces, which consists of the direct visualization and identification of the parasites eliminated through the feces. These tests are generally low sensitive and the microscope slides contain a large number of impurities, which can impair the result of the diagnosis. In order to improve the diagnostic accuracy, a new parasitological technique called Three Fecal Test (TF‑Test) was developed. To further improve its diagnostic accuracy, few modifications of the original protocols have been made with the years. In this study the performance of these new techniques to detect gastrointestinal parasites in human and animal fecal samples was described and discussed in relation to the performance of other conventional coprological tests. It could be concluded that the TF‑Test conventional and modified can be used for the diagnosis of several human and animal parasites, with satisfactory results.</p> Sandra Valéria Inácio Jancarlo Ferreira Gomes Alexandre Xavier Falcão Débora Regina Romualdo da Silva Walter Bertequini Nagata Bianca Martins dos Santos Felipe Augusto Soares Saulo Hudson Nery Loiola Aline do Nascimento Benitez Stefani Laryssa Rosa Katia Denise Saraiva Bresciani Copyright (c) 2022 Veterinaria Italiana 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2040.10928.1 An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats <p>The Orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry‑over contamination associated with LAMP as open tube format prevents the assay applicability as point‑of‑care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre‑addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO<sub>4</sub> for these dyes, respectively. This optimized assay that could reveal the result within one hour is highly specific and sensitive with a limit of detection of 12.5 femtogram of viral genomic DNA or ~ 85 virus genome equivalent. This improved method can also prevent the cross‑contamination of LAMP reactions in the laboratory without compromising diagnostic sensitivity and specificity when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.</p> Gnanavel Venkatesan Anand Kushwaha Amit Kumar D.P. Bora P. Sasikumar Copyright (c) 2022 Gnanavel Venkatesan 2022-12-30 2022-12-30 58 2 10.12834/VetIt.2426.15340.2