Short communication: Parapoxvirus and Orthopoxvirus coinfection in milk of naturally infected cows.

Several studies have shown the occurrence of poxvirus infections associated with exanthematic lesions in cattle from many Brazilian states. Coinfection between viruses belonging to 2 genera, Orthopoxvirus (OPXV) and Parapoxvirus (PPV), was already identified from the lesions of affected cows and humans. The DNA and infectious viral particles of Vaccinia virus, an OPXV, have been detected in milk of naturally and experimentally infected cows. However, to date no reports have described the detection of Pseudocowpox virus, a PPV, in milk. Thus, we investigated the presence of PPV and OPXV in milk samples obtained from dairy cows from a Brazilian region with exanthematic disease outbreaks. From 2011 to 2014, 6 dairy farms with exanthematic disease outbreaks involving dairy cows, calves, and humans were visited. Twelve crusts of cows' teat lesions and 60 milk samples were collected. The crusts and milk samples were analyzed by PCR to detect OPXV or PPV DNA. According to the analyzed crusts, we detected PPV infection in 4 of the 6 visited farms, from which we investigated the PPV contamination in milk. From the 40 milk samples tested, PPV DNA was detected in 12 samples. Of these milk samples, 8 were positive for both PPV and OPXV. This is the first report of PPV DNA detection in milk samples from affected cows, indicating that the virus may be present in milk and potentially contaminating dairy products associated or not with OPXV. In addition to the lesions caused by direct contact, the presence of 2 or more poxvirus species in milk showed that the effect of zoonotic exanthematic diseases on public health and animal husbandry is relevant and cannot be overlooked.

Short communication: Survival of Vaccinia virus in inoculated cheeses during 60-day ripening.

Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 102 pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d.

Clinical, hematological and biochemical parameters of dairy cows experimentally infected with Vaccinia virus.

Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an important zoonosis that affects dairy cattle. There are many aspects of the disease that remain unknown, and aiming to answer some of these questions, the clinical, hematological, and biochemical parameters of VACV experimentally infected cows were evaluated. In the first part of the study, lactating cows were infected with VACV-GP2 strain. In the second part, animals previously infected with VACV-GP2 were divided into two treatment groups: Group 1, immunosuppressed cows; and Group 2, re-infected cows. In this study, BV could be experimentally reproduced, with similar lesions as observed in natural infections. Moreover, a short incubation period and local lymphadenopathy were also observed. VACV could be detected by PCR and isolated from scabs taken from teat lesions of all inoculated and re-inoculated animals. Lymphocytosis and neutrophilia were observed in all animals from the first part of the experiment, and lymphopenia and relative neutrophilia were observed in the immunosuppressed animals. Detection of viral DNA in oral mucosa lesions suggests that viral reactivation might occur in immunosuppressed animals. Moreover, clinical disease with teat lesions may occur in previously VACV-infected cows under the experimental conditions of the present study.

Bovine vaccinia, a systemic infection: evidence of fecal shedding, viremia and detection in lymphoid organs.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that affects dairy cattle and milkers, causing economic losses and impacting animal and human health. Based on the clinical presentation, BV appears to be a localized disease, with lesions restricted to the skin of affected individuals. However, there are no studies on the pathogenesis of the disease in cows to determine if there is a systemic spread of the virus and if there are different ways of VACV shedding. The objective of this work was to study if there is a systemic spread of VACV in experimentally infected cows and to study the kinetics of VACV circulation in the blood and shedding in the feces of these animals. To this end, eight crossbred lactating cows were used. Three teats of each cow were inoculated with the GP2V strain of VACV. All animals were monitored daily, and blood and fecal samples were collected for 67 days post-infection (dpi). After this period, four of these previously infected cows were immunosuppressed using dexamethasone. Viral DNA was continuously detected and quantified in the blood and feces of these animals in an intermittent way, even after the resolution of the lesions. At slaughter, tissues were collected, and viral DNA was detected and quantified in the mesenteric and retromammary lymph nodes, ileum, spleen and liver. The detection of VACV DNA in the feces for a longer period (67 dpi) and in the lymphatic organs provides new evidence about VACV elimination and suggests that BV could be a systemic infection with a chronic course and viral shedding through the feces.

Vaccinia virus is not inactivated after thermal treatment and cheese production using experimentally contaminated milk.

Bovine vaccinia is an emergent zoonosis caused by the Vaccinia virus (VACV). The disease is characterized by the appearance of exanthematic lesions that occur in humans and dairy cows. Previous studies have revealed the presence of infectious viral particles in milk samples during an outbreak of bovine vaccinia in Brazil, indicating the possibility of disease transmission through raw milk. To assess the viability of the virus in milk after thermal treatment and processing procedures, milk samples were experimentally contaminated with 10(3) plaque forming units (PFU)/mL (group I) and 10(5) PFU/mL (group II) VACV Guarani P2 virus, and the third group was not contaminated and served as a control. The samples were submitted to storage temperatures in a cold chamber, freezer for 48 hours, and to low temperature long-time treatment. Moreover, the viral viability was evaluated in cheese produced with contaminated milk using 10(4) PFU/mL VACV Guarani P2. Notably, the virus remained viable in milk after storage for 48 hours in both the cold chamber and the freezer, with a reduction in viral titer of 14.49% and 25.86%, respectively. Group II showed a viral reduction in titer of 61.88% and 75.98%, respectively. Thermal treatment 65°C for 30 minutes showed a reduction of viral titer of 94.83% and 99.99%, respectively, in group I and group II, but still showed remaining viable virus particles. In addition, it was possible to recover infectious viral particles from both the solid curds and the whey of the cheese produced with experimentally contaminated milk. The cheese shows a reduction in viral titer of 84.87% after storage at 4°C for 24 hours. The presence of viable viral particles in milk after both thermal treatment and cheese production indicates a potential public health risk.