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SARS-CoV-2 was a worldwide threat during the COVID-19 pandemic, and the state of Mato Grosso had the second highest mortality rate in Brazil, with 427. 4 deaths/100,000 inhabitants. However, no large-scale study among dogs and cats in such highly infected areas of Brazil has so far been conducted. Accordingly, the present study reports on a serosurvey among dogs and cats in Cuiabá, capital of Mato Grosso from November 2020 to July 2021, where the human mortality rate was 605/100,000 at that time. Overall, 33/762 dogs (4.3%) and 4/182 cats (2.2%) were found to be seropositive for SARS-CoV-2 through ELISA, and 3/762 dogs (0.4%) and 3/182 cats (1.6%) were seropositive through the serum neutralization test. Cats presented higher seroprevalence with higher titers of neutralizing antibodies. Although N-protein based ELISA may be a good screening test, cross-reactivity with other canine coronaviruses may impair its diagnostic use among dogs.

West Nile virus (WNV) is an important zoonotic Flavivirus responsible for mild fever to severe neurological disease in humans and horses. Despite the occurrence of major previous outbreaks in Namibia and the likelihood of the current endemicity of the virus, only limited investigations and monitoring activities of WNV have been performed in the country. The use of animal sentinels is a valuable approach toward investigating the infection presence in an area and to predict the potential occurrence of human outbreaks. Serological investigations in dogs hold several advantages, considering their infection susceptibility, the ease of sample handling, and the evaluation of risk factors of pet owners that share the same habit with their pets. To evaluate the usefulness of such a sero-epidemiological investigation in Namibia, a broad serosurvey was performed in 2022 that included 426 archived domestic dog samples from eight Namibian regions. Although the ELISA prevalence, indicative of Flavivirus infection, was relatively high (16.43%; 95 CI: 13.10–20.39%), the virus neutralization test confirmed only a minority of cases, highlighting a prevalence of 2.82% (95 CI: 1.47–4.90%), significantly lower than in Namibian donkeys and reports from other countries. Variables that could explain the recorded differences remain to be explored, including animal exposure, variable vector presence, distribution, and feeding preferences. The study results suggest the limited usefulness of dogs as sentinels for WNV monitoring in Namibia.

Bluetongue virus (BTV) is the etiologic agent of bluetongue (BT), a viral WOAH-listed disease affecting wild and domestic ruminants, primarily sheep. The outermost capsid protein VP2, encoded by S2, is the virion’s most variable protein, and the ability of reference sera to neutralize an isolate has so far dictated the differentiation of 24 classical BTV serotypes. Since 2008, additional novel BTV serotypes, often referred to as “atypical” BTVs, have been documented and, currently, the full list includes 36 putative serotypes. In March 2015, a novel atypical BTV strain was detected in the blood of asymptomatic goats in Sardinia (Italy) and named BTV-X ITL2015. The strain re-emerged in the same region in 2021 (BTV-X ITL2021). In this study, we investigated the pathogenicity and kinetics of infection of BTV-X ITL2021 following subcutaneous and intravenous infection of small ruminants. We demonstrated that, in our experimental settings, BTV-X ITL2021 induced a long-lasting viraemia only when administered by the intravenous route in goats, though the animals remained healthy and, apparently, did not develop a neutralizing immune response. Sheep were shown to be refractory to the infection by either route. Our findings suggest a restricted host tropism of BTV-X and point out goats as reservoirs for this virus in the field.

SARS-CoV-2 has been shown to lose the furin polybasic cleavage site (FCS) following adaptation on cell culture. Deletion occurring in this region, which may include also the FCS flanking regions, seem not to affect virus replication in vitro; however, a chimeric SARS-CoV-2 virus without the sole FCS motif has been associated with lower virulence in mice and lower neutralization values. Moreover, SARS-CoV-2 virus lacking the FCS was shed to lower titers from experimentally infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. In this study, we investigated the replication kinetics and cellular tropism of a SARS-CoV-2 isolate carrying a 10-amino acid deletion in the spike protein spanning the FCS in lung ex vivo organ cultures of mink. Furthermore, we tested the neutralization capabilities of human convalescent SARS-CoV-2 positive serum samples against this virus. We showed that this deletion did not significantly hamper neither ex vivo replication nor neutralization activity by convalescent serum samples. This study highlights the importance of the preliminary phenotypic characterization of emerging viruses in ex vivo models and demonstrates that mink lung tissues are permissive to the replication of a mutant form of SARS-CoV-2 showing a deletion spanning the FCS. Notably, we also highlight the need for sequencing viral stocks before any infection study as large deletions may occur leading to the misinterpretation of results.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved rapidly, leading to viral lineages characterized by multiple mutations in the spike protein, which could potentially confer to the virus the ability to avoid the vaccine-induced immune response, making the vaccines less effective or ineffective. Here, we initially evaluated the neutralization capabilities in vitro by serum neutralization (SN) of six serum samples collected from recipients of the BNT162b2 vaccine against 11 SARS-CoV-2 isolates belonging to the major SARS-CoV-2 lineages that had been circulating in Italy. Then, we considered 30 additional serum samples by SN assay against the dominant B.1.617.2 (Delta) variant. A B.1 lineage isolate was used as a reference. In the first analysis, significant differences when compared with the reference strain (p > 0.05) were not evidenced; instead, when the panel of 30 sera was tested against the B.1.617.2 (Delta) variant, a significant (p = 0.0015) 2.38-fold reduction in neutralizing titres compared with the reference after the first vaccine dose was demonstrated. After the second vaccine dose, the reduction was not significant (p = 0.1835). This study highlights that the BNT162b2 vaccine stimulates a humoral response able to neutralize all tested SARS-CoV-2 variants, thus suggesting a prominent role in mitigating the impact of the SARS-CoV-2 pandemic in real-world conditions. Long-term follow-up is currently ongoing.

In Zimbabwe, there have been no chlamydiosis and limited brucellosis studies in goats. This study was conducted to determine the seroprevalence and risk factors of the two diseases in goats at three different livestock–wildlife interface areas: porous, non-porous and non-interface in the south-eastern lowveld of Zimbabwe. Collected sera (n = 563) were tested for Brucella antibodies using the Rose Bengal plate test (RBPT) and the complement fixation test (CFT); and for Chlamydia abortus antibodies using the CFT. All tested goats were negative for Brucella antibodies. Overall, chlamydial seroprevalence was 22%. The porous [c2 = 9.6, odds ratio (OR) = 2.6, p = 0.002] and non-porous (c2 = 37.5, OR = 5.8, p < 0.00001) interfaces were approximately three and six times more likely to be chlamydial seropositive than the non-interface area, respectively. Chlamydial seroprevalence was not associated with sex (c2 = 0.5, OR = 1.2, p = 0.5), abortion history in female goats (c2 = 0.7, OR = 1.3, p = 0.4), keeping goats with cattle (c2 = 0.2, OR = 1.5, p = 0.7) or flock size (c2 = 0.03, OR = 1.4, p = 0.9). Our study provides the first serological evidence of chlamydiosis in goats in Zimbabwe and the results suggest that proximity to wildlife is associated with increased chlamydial seropositivity. Further studies are required to determine the role of chlamydial infection on goat reproductive failure and that of wildlife on C. abortus transmission to domestic ruminants.

By late March 2020, Villa Caldari, a small village of the municipality of Ortona (Abruzzo region), was registering an incidence rate of COVID-19 cases ten times greater than the overall municipality and was declared a hotspot area. Twenty-two days later, epidemiological investigation and sampling were performed, to evaluate SARS-CoV-2 circulation and the presence of SARS-CoV-2 antibodies. Overall, 681 nasopharyngeal swabs and 667 blood samples were collected. Only one resident of the village resulted in being positive for RNA viral shedding, while 73 were positive for SARS-CoV-2 antibodies. The overall seroprevalence was 10.9%. The difference between the seroprevalence of infection in asymptomatic and symptomatic individuals was significant (χ2 = 14.50 p-value = 0.0001). Amongst the residents positive for antibodies, fatigue and/or muscle pain, fever and anosmia were the most experienced symptoms, whose most frequent onset was observed during the first two weeks of March. Familial and habit-related clusters were highlighted. Nevertheless, the investigations showed a low SARS-CoV-2 circulation in the village at the time of the sampling, demonstrating virus transmission could be limited when strict emergency measures are followed. Given the favorable results, the emergency measures were then lifted.

Ovine sera collected from the northern Prefectures of Hokkaido, Iwate and Aomori in Japan, were examined for the presence of antibodies against Coxiella burnetii (Q fever) using the complement fixation test and, against Brucella spp., using both the rapid serum agglutination test and the complement fixation test. None of the sera tested were serologically positive to Brucella spp. A total of 21 animals (8.64%) out of 243 samples tested were seropositive to the C. burnetii antigen. Levels of infection were observed in all of the three Prefectures and in ten flocks of the fourteen sampled. Although no diagnostic measures were in place, the infection could not be linked to losses in sheep production or to the decreased fertility in ewes, a lower lambing rate and mortality in lambs. These data confirmed that Q fever is widespread in the sheep population in the area studied. Considering the zoonotic potential of the disease, further studies to investigate the epidemiology of Q fever in this region are required.

Sono stati sviluppati una ELISA competitiva (c-ELISA), una ELISA indiretta (i-ELISA) e un test immunologico DELFIA (Dissociation-Enhanced Lanthanide Fluorescence Immunoassay) per la ricerca di anticorpi verso Brucella suis in sieri di maiale e cinghiale. I tre test prevedono l’utilizzo di un anticorpo monoclonale (MAb 4B5A) verso l’LPS di Brucella (c-ELISA e DELFIA) e di un anticorpo monoclonale (MAb 10C2G5) verso le IgG suine (i-ELISA). La specificità (Sp) e la sensibilità (Se) dei tre test sono le seguenti: per la c-ELISA Se e Sp = 100% con un valore di cut-off pari al 61.0% (B/B0%); per la i-ELISA Sp = 99.1% e Se = 100% con un valore di cut-off di 21.7% (PP%); per il DELFIA Sp = 91.0% e Se = 75% ponendo il valore di cut-off al 37.0% (B/B0%). Inoltre sono state valutate le performance, nei confronti di sieri suini, di un test FPA (Fluorescence Polarization Assay) commerciale sviluppato per la ricerca di anticorpi anti-Brucella in sieri bovini; la specificità e la sensibilità ottenute sono entrambe del 100% al valore di cut-off di 99.5 (mP). Questi risultati suggeriscono che la combinazione di c-ELISA, i-ELISA e FPA può essere utilizzata per migliorare la diagnosi di brucellosi suina.

A competitive enzyme-linked immunosorbent assay (c-ELISA), an indirect ELISA (i-ELISA) and a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) were developed to test for antibodies to Brucella suis in pig and wild boar sera. An anti-Brucella-LPS monoclonal antibody (MAb 4B5A) (c-ELISA and DELFIA) and an anti-swine IgG monoclonal antibody (MAb 10C2G5) (i-ELISA) were used for the three assays. The specificity (Sp) and sensitivity (Se) of the assays gave the following results: Se and Sp = 100% at a cut-off value of 61.0% (B/B0%) for c-ELISA; Sp = 99.1% and Se = 100% at a cut-off value of 21.7% (percentage positivity: PP%) for i-ELISA; Sp = 91.0% and Se = 75% at a cut-off value of 37.0% (B/B0%) for DELFIA. In addition, the performance of a commercial fluorescence polarisation assay (FPA), standardised for bovine sera, was evaluated in swine sera. The specificity and sensitivity obtained were both 100% at a cut-off value of 99.5 (millipolarisation unit values). These results suggest that the combination of c-ELISA, i-ELISA and FPA can be used to improve the serological diagnosis of swine brucellosis.

È stata valutato un test ELISA indiretto per rilevare la presenza di anticorpi brucellari nel latte di bufala (Bubalus bubalis Linnaeus, 1758). L’accuratezza della prova è stata valutata su latte proveniente da allevamenti bufalini campani. Sono stati impiegati 100 campioni negativi provenienti da dieci allevamenti ufficialmente indenni della provincia di Salerno e 30 campioni positivi provenienti da tre allevamenti della provincia di Caserta con animali positivi ai test ufficiali e presenza di Brucella abortus biovar 1. La sensibilità della prova è stata del 100% (intervallo di confidenza [IC] del 90,8%-100%) e la specificità sui campioni di latte individuale è stata del 98% (IC 93%-99,4%). Al fine di simulare campioni di latte di massa provenienti da allevamenti infetti a varie prevalenze di infezione, sono state analizzate diluizioni da 1:10 a 1:100 di campioni di latte positivo in latte negativo. La probabilità di rilevare anticorpi in campioni di latte positivo è risultata superiore al 50% fino alla diluizione 1:50 in latte negativo. Considerando la consistenza media nazionale degli allevamenti bufalini, la probabilità di individuare l’infezione in un allevamento, ricorrendo alla prova ELISA su latte di massa, è risultata del 50% (IC 33,1%-66,9%) qualora nel latte di massa fosse presente il latte di un solo animale infetto.

An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated for the detection of Brucella antibodies in milk from water buffalo (Bubalus bubalis Linnaeus, 1758). The test accuracy was evaluated on milk samples from the Campania Region in Italy. A total of 100 negative samples were collected from 10 officially brucellosis-free herds in Salerno Province, while 30 positive samples were collected from 3 herds in Caserta Province, where animals gave positive results to the official tests and it was here that Brucella abortus biovar 1 had been isolated. Test sensitivity was 100%, with a confidence interval (CI) of 90.8%-100%, while specificity was 98% (CI 93%-99.4%) on individual milk samples. To simulate bulk milk samples from herds infected at various levels of infection, dilutions from 1:10 to 1:100 of positive milk samples in negative milk were also used. The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:50 in negative milk. Considering the average national water buffalo herd size, the probability of identifying infection in a water buffalo herd by bulk milk testing is 50% (CI 33.1%-66.9%) in the worst case scenario of a single infected animal contributing to the bulk milk.

Il patrimonio zootecnico della specie bufalina (Bubalus bubalis) della regione Campania, è di 250 000 capi, di questi 150 000 allevati in aziende zootecniche della provincia di Caserta. In queste aziende, nel 2007, l’infezione da Brucella abortus ha avuto la prevalenza media, per allevamento, del 20%. Complessivamente, i 2/3 degli allevamenti positivi hanno evidenziato una prevalenza superiore al 10% e, di questi, i 3/4 una prevalenza superiore al 20%. Prendendo il 20% come valore di riferimento, la metà degli allevamenti infetti (22% degli allevamenti casertani) ha evidenziato prevalenze inferiori o uguali al 20%, la restante metà (un altro 22% del totale) prevalenze comprese tra il 20 e il 56%. In questo contesto epidemiologico è stato adottato un piano di eradicazione della brucellosi che prevedeva l’abbattimento dei capi infetti e la vaccinazione del restante patrimonio bufalino delle zone con più alta incidenza. Per la profilassi vaccinale della brucellosi, il Manual of diagnostic tests and vaccines for terrestrial animals (OIE) prevede l’utilizzo del vaccino B. abortus S19 (S19). Purtroppo, l’utilizzo del vaccino negli animali adulti non è privo di possibili effetti indesiderati. Per superare questo aspetto negativo è stato ipotizzato l’impiego del vaccino di B. abortus RB51 (RB51) anche se in letteratura scientifica, sono risultati disponibili pochi dati relativi alla corretta dose vaccinale, all’efficacia e all’innocuità del vaccino nel bufalo. A tale scopo è stato condotto uno studio comparativo tra i due vaccini. Sono state utilizzate 13 femmine di bufalo di 5 mesi di età provenienti da un allevamento ufficialmente indenne da brucellosi. Un gruppo di 5 animali è stato vaccinato due volte, a distanza di un mese, con una dose di RB51 tre volte superiore a quella prevista per i bovini; un secondo gruppo di 5 bufale con S19 rispettando il dosaggio raccomandato per i bovini e un terzo gruppo, di 3 animali di controllo, inoculato con tampone fosfato. Trenta giorni dopo la vaccinazione è stato effettuato un inoculo con ceppo virulento B. abortus 544. Gli anticorpi sviluppati nei 5 animali vaccinati con SRB51 non sono stati rilevati mediante sieroagglutinazione rapida con antigene al Rosa Bengala (SAR) o fissazione del complemento (FdC). Gli stessi animali sono stati sottoposti a test FdC allestito con antigene specifico RB51 (FdC-RB51). Dopo l’abbattimento, sono stati prelevati milza e linfonodi retrofaringei e sopramammari per la diagnosi diretta di brucellosi. Per comparare l’immunogenicità ottenuta nei bufali dei tre gruppi sperimentali è stata effettuata la valutazione statistica.

Approximately 250 000 water buffalo (Bubalus bubalis) live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150 000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51) has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19). The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT) and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group.

The results of an enzyme-linked immunosorbent assay (ELISA) implemented for the detection of gamma interferon (g-interferon) production in cattle vaccinated with Brucella abortus strain RB51 are presented. A purified protein fraction derived from RB51 (RB51 brucellin) has been used as antigenic stimulus for whole blood. The test was evaluated for 300 days in ten heifers vaccinated at calfhood with 10 × 109 colony-forming units of RB51 and in five control heifers. All animals came from officially brucellosis-free herds. Vaccinated animals started to give positive results from day 17 post vaccination (pv) until day 239 pv. All vaccinated animals gave a positive reaction at least once (with a stimulation index exceeding 2.5). Nevertheless, if sampling on day 20 pv is excluded (90% of vaccinated animals gave positive results), the sensitivity of the test varies from 20% to 70%, with a 40% average. A stimulation index over 2.5 was also recorded in three control animals. The results suggest that the g-interferon test is not suitable for the detection of cattle vaccinated with RB51, either at the individual or at the herd level.

In questo lavoro sono presentati i risultati di un test ELISA messo a punto per rilevare la produzione di gamma interferone (g-interferone) in bovini vaccinati con Brucella abortus ceppo RB51 (RB51). Come stimolo antigenico per il sangue intero è stata utilizzata una frazione proteica purificata derivante da RB51 (brucellina RB51). La prova è stata valutata nell’arco di 300 giorni in 10 manze vaccinate in età prepubere con 10×109 Unità Formanti Colonia di RB51 e in cinque manze di controllo, provenienti da allevamenti ufficialmente indenni da brucellosi bovina. I capi vaccinati hanno cominciato a fornire risultati positivi a partire dal 17° giorno post vaccinazione (p.v.) fino al giorno 239 p.v. Tutti i capi vaccinati hanno fornito almeno una volta un risultato positivo (indice di stimolazione, IS, superiore a 2,5). Tuttavia, se si esclude il prelievo al giorno 20 p.v. (90% di animali vaccinati risultati positivi), la sensibilità del test oscilla tra il 20% e il 70%, con una media del 40%. IS superiore a 2,5 è stato rilevato anche in tre animali di controllo. Sulla scorta dei risultati ottenuti, si ritiene che il test del g-interferone non fornisce garanzie sufficienti per consigliarne l’impiego ai fini di riconoscere i bovini vaccinati con RB51, sia come prova individuale, sia come prova d’allevamento.

I metodi ufficiali previsti dal Piano nazionale di eradicazione della brucellosi bovina ed ovi-caprina sono la sieroagglutinazione rapida (SAR) con l’antigene acidificato al Rosa Bengala e la fissazione del complemento (FDC). Nella attuale fase del piano di eradicazione non è infrequente imbattersi in risultati di difficile interpretazione ottenuti con i test ufficiali, pertanto è necessario poter disporre di test aggiuntivi che presentino livelli di specificità e di sensibilità più elevati. A questo scopo sono stati validati due metodi ELISA, indiretto (i-ELISA CL) e competitivo (c-ELISA CL), mediante l’utilizzo di un substrato chemiluminescente per la determinazione di anticorpi anti-Brucella in siero bovino ed ovino. I metodi si basano sulla rivelazione degli anticorpi anti-Brucella, contenuti nel siero, mediante la catalisi di un substrato enzimatico chemiluminescente (sistema luminolo/H2O2/ enhancer), da parte della perossidasi coniugata ad anticorpi secondari anti-IgG, nella i-ELISA CL, o al monoclonale anti-LPS, nella c-ELISA CL. Sulla base dei risultati ottenuti, per l'i-ELISA CL è stato stabilito un valore di cut-off, espresso come percentuale di positività (PP), del 60% per i sieri bovini e del 37,5% per quelli ovini; con questo valore di cut-off si ottiene una sensibilità ed una specificità del test del 100% per i sieri bovini, e una sensibilità del 100% e una specificità del 99,8% per quelli ovini. Per la c-ELISA CL è stato scelto un cut-off, espresso come percentuale di inibizione (PI), del 30% per i sieri bovini e del 40% per quelli ovini, che assicura valori di sensibilità e specificità del 100% in entrambi i casi.

The official methods specified in the national bovine and ovine/caprine brucellosis eradication plan are the Rose Bengal and complement fixation tests. In the current phase of the eradication plan, it is often difficult to interpret the results obtained with the official tests. Consequently, additional tests that offer greater sensitivity and specificity are thus required. For this reason, two methods, the indirect chemiluminescence enzyme-linked immunosorbent assay (i-ELISA CL) and the competitive chemiluminescence ELISA (c-ELISA CL) that use a chemiluminescent substrate to determine anti-Brucella antibodies in bovine and ovine serum were validated. The methods are based on the detection of anti-Brucella antibodies in serum by catalysis of a chemiluminescent enzyme substrate (luminol/ H2O2/enhancer system) by peroxidase conjugated to secondary anti IgG antibodies in i-ELISA CL and to monoclonal anti-lipopolysaccharide (LPS) antibodies in c-ELISA CL. From the results obtained, a cut-off of 60% for bovine serum and 37.5% for ovine serum, expressed as positivity rate (PR), were established Using these cut-off values, for the i-ELISA CL, 100% sensitivity and specificity was obtained for bovine serum and 100% sensitivity and 99.8% specificity for ovine serum. Cut-off values of 30% for bovine serum and 40% for ovine serum, expressed as inhibition rate, were selected for c-ELISA CL, which ensured 100% sensitivity and specificity in both cases.

Immune response to Brucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv), the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv). Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI) 76.2%–100%). The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF) areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated animals.