Bovine Coronavirus Updates

Jeremy Schefers DVM, PhD Minnesota Veterinary Diagnostic Labortory Take-home messages

1. Serum antibody titers, from either vaccination or natural exposure, don’t appear to last longer than 6 months. 2. BoCV infection appears to compromise the innate mucociliary apparatus of upper respiratory epithelium. Bacterial and viral pathogens in diseased bovine lung, 2015

45% 42% Bovine Coronavirus was detected in 40% 38% 25% of diseased cattle lung.

35%

30% 25% 25% Blue bars are bacteria 20% 18% Red bars are viruses 15%

10% 8%

5% 2% 1% 0% Histophilus Pasteurella Bovine Mannheimia Bovine Infectious Parainfluenza 3 somni multocida Coronavirus hemolytica Respiratory Bovine Syncytial Rhinotracheitis 3 2016 BoCV Goals • Immunity – Detect seronegative populations – Determine titers generated by vaccination – Determine titers generated by natural infection – Estimate decay of serum antibody titer • Pathology of infection – Examine extent of virus infection – Determine the impact of virus on upper respiratory (head) tissues Seropositive dairy cows

Lactating Holstein cows developed winter dystentery in April.

11 Cows sampled in late July 2 cows = neg 5 cows = 160 3 cows = 320 1 cow = 640 Seropositive dairy calves

July 2016

1-week-old Holstein calves fed colostrum from vaccinated dams

3 calves bled: Calf 1 = 640 Calf 2 = 320 Calf 3 = 320 Partially seronegative heifers August 2016

5-month-old Holstein heifers

5 bled: 3 = seronegative 2 = titers of 80.

These calves were vaccinate with Calf Guard at birth and fed colostrum from vaccinated dams. Seronegative dairy heifers

July 2016

Yearling Holstein heifers

6 bled and all six seronegative for BoCV (titer <20). Seronegative springing heifers

July 2016

Pregnant Holstein heifers due to calve in fall

6 bled and all seronegative for BoCV (titer <20). Dairy herd serological profile

Sample Description Result Titer 1 Lactating Heifer "Aim" Not vacc POS 160 2 Lactating Heifer 1327 Not vacc POS 320 3 Lactating Heifer "Tahiti" Not vacc Neg <20 4 Lactating Heifer "Sansa" Not vacc POS 160 Lactating cows: 5 Lactating Heifer "Icon" Not vacc POS 160 6 Dry Cow "Now" Vacc 12/10/15 POS 320 Exposed, diseased, titer 7 Dry Cow "Vegas" Vacc 12/7/2015 POS 160 mean of 160, 3 months 8 Dry Cow "Concord" Vacc 12/12/2015 POS 160 9 Dry Cow "Savanna" Vacc 12/6/2015 Neg <20 post winter dysentery 10 Dry Cow "Claret" Vacc 12/15/2015 POS 320 11 Dry Cow "Motor" Vacc 12/8/2015 POS 640 12 Newborn calf 4 POS 640 3 newborn calves: 13 Newborn calf 5 POS 320 14 Newborn calf 6 POS 320 Colostral antibody titers > 320. 15 6-month-old heifer - 1390 Neg <20 16 6-month-old heifer - 1392 POS 80 17 6-month-old heifer - 1393 Neg <20 18 6-month-old steer - 825 POS 80 Heifers >6 months: 19 6-month-old steer - 826 Neg <20 20 Yearling heifer 1364 lot 1 Neg <20 Antibody titers decay away by 21 Yearling heifer 1365 lot 1 Neg <20 6 months and cattle remain 22 Yearling heifer 1367 lot 1 Neg <20 23 Yearling heifer 1368 lot 1 Neg <20 negative over summer. 24 Yearling heifer 1369 lot 1 Neg <20 25 Yearling heifer 1371 lot 1 Neg <20 BoCV titers in two large dairy herds October 2016 Holstein cows developed winter dystentery & respiratory disease every winter. Vaccinated with killed BoCV scour vaccine at dry off.

Herd 1 (n=12) 5 = Negative 2 = 80 5 = 160

Herd 2 (n=8) 2 = Negative 2 = 40 2 = 80 2 = 160 Seronegative cows in vaccinated, lactating dairy herds. Oct 2016 • By fall, 25 – 42% of cows in 2 herds were seronegative for BoCV. • It appears that is enough naïve cows to compromise herd immunity. • Most of those cattle are more than 120 DIM (6+ months from dry cow vacc.) and less than 220 DCC (dry off). • In the fall, one farm vaccinated (IM MLV) all cows 120+ DIM and less that 220 DCC. • To date, there have been no episodes of winter dysentery or widespread respiratory disease.

Seronegative beef calves

July 2016

3-month-old Angus calves

6 bled: All seronegative

Neither the calves or dams are vaccinated for BoCV Pasture behavior considerations

Comfortable and content cattle spread out. Nose-to- nose contact is limited.

Uncomfortable cattle concentrate under trees and at water source to alleviate heat & fight flies. Risk for pathogen shedding and transmission among naïve calves is increased. 3-month-old dairy steers July 2016

IM vaccinated at 1 & 5 weeks with MLV BoCV while in calf hutches.

6 calves sampled: All 6 had titers of 320. Do serum antibody titers matter? Day 5 BoCV Ab titer of fatal pneumonia cases and asymptomatic cohorts 1200 (Storz, Journal of Clinical Microbiology, 2000)

1000

Asymptomatic = These cattle were 800 18 normal controls that had titers, remained clinically healthy, and did not nasally shed BCoV 600 Red circles = Mortalities

These cattle died and antibody titer antibody

Fatality had very low / absent Asymptomatic

400 BCoV antibody titers on BoCV day 5 200

0 0 10 20 30 40 Animal BoCV in a feedlot (Hasoksuz, JVDI, 2002)

• Ohio Feedlot Study – Peak of BoCV nasal and fecal shedding occurred at 4 days post arrival – Day 1: 11% had antibody titers against BoCV – Day 21: 91% had antibody titers against BoCV – Seroconversion was inversely related to average daily weight gains (p < 0.06) • BoCV is highly contageous and affected gain when naïve animals seroconverted. BoCV respiratory infection on health and performance (Lathrop, AJVR, 2000) 837 cattle, 4 feedlots, 2 states (Ohio and Texas)

IN Shed BoCV Serology Outcome Yes Seronegative on 1.6X more likely to be treated than arrival non-shedding, seropositive cattle No Seronegative Slightly higher risk to be treated on arrival Yes 2.2X more likely to have pulmonary lesions at harvest

• In this study, neither BoCV shedding or seroconversion were predictors of average daily gain (ADG) • In another study, seropositive cattle gained 13 lbs. more than seronegative cattle. (Hasoksuz, JVDI, 2002)

Vaccine study

• West pens 1 – 4: 80 calves = ½ dose of SQ killed BoCV at 5 weeks • East pens 7-10: 80 calves = Intranasal MLV BoCV at 5 weeks • Middle pens 5&6: 40 Non-vaccinated controls Vaccine study results

• <5% morbidity • No mortalities • Weighed at 5 months • SQ Killed BoCV = 399 lbs. • IN Mod LV BoCV= 385 lbs • Neg controls = 373 lbs • Significance? – Killed SQ vs. Controls p =0.01 – ML Intranasal & Controls p=0.20 Updated vaccine study

<5% morbidity No mortalities Weighed at 5 months

IM MLV BoCV = 425 lbs. SQ Killed BoCV = 399 lbs. IN MlV BoCV= 385 lbs. Neg controls = 373 lb. BoCV Nasal Swab Monitoring

Date Test result April 2011 (no vaccine) Positive, Ct 30 August 2011 (vaccinated with killed IM) Positive, Ct 25 March 2013 (vaccinated with killed IM) Positive , Ct 30 March 2013 (vaccinated with killed IM) Positive, Ct 34 in 1/4 calves June 2013 (switched to IM Calf Guard) negative October 2013 (continued IM Calf Guard) negative June 2014 (continued IM Calf Guard) negative March 2015 (continued IM Calf Guard) negative Vaccination expectations Positive impact – Dairy calves BEFORE grouping (hutch to pen) Unlikely vaccine impact – Beef calves BEFORE – Young dairy calves on bunching on pasture autofeeders. Exposure is and/or weaning immediate & overwhelming. – Mid to late lactation dairy – Upon feedlot arrival. cows BEFORE winter Exposure is immediate and high. – Show cattle BEFORE shows* – In the face of disease. Exposure is overwhelming. Some research has shown *Cold water wash BoCV is not affected by racks appear to interferon. facilitate transmission at shows (personal observation) BoCV: Pathology of infection and diagnosis Bovine coronavirus Fragile, host-derived lipid envelope. 1. Susceptible to heat, UV light and disinfectants 2. Envelope needs to remain intact for entry into cells. 3. Surface antigens or “keys” can be covered and neutralized by BoCV antibodies. 4. Diagnosis - Virus needs to be “fresh” and remain intact for detection by electron microscopy. Dead, degraded virus is easily detected by PCR. 5. Virus isolation (growth) is challenging. Diagnosis - Comparing EM and PCR on 300 fecal sample from calves

80% 70% In these fecal samples, PCR 70% was 5X more sensitive than

60% EM.

50% 43% 43%

40%

Percent 30%

20% PCR

10% 8%

0% EM Rotavirus Coronavirus EM PCR Calf diarrhea, the most lethal syndrome of BoCV: Acute mucohemorrhagic enterocolitis leading to bicarbonate loss, metabolic acidosis and cardiotoxic hyperkalemia.

Calf colon IHC: Necrotic colitis Upper respiratory BoCV: Histopathology & IHC

H & E histopathology: Attenuated epithelium. Nasal turbinate Trachea H&E and IHC H&E and IHC Is BoCV a respiratory pathogen? If so, what is the lesion? IBRV – Erosions, ulcers and necrosis with intranuclear inclusion bodies. BRSV – Syncytial cells lining airways with intracytoplasmic inclusion bodies. PRRSV – Interstitial pneumonia and necrotizing alveolitis. SIV – Necrotizing bronchiolitis. Bovine coronavirus –????. Case 1 • Beef calf developed summer pneumonia in late July and died. • Trachea  • BoCV was found on a nasal swab, in the sinus cavity, facial nerve, retropharyngeal & submandibular lymph nodes, salivary gland and BoCV in trachea (IHC) olfactory (smell) nerve Case 2 – Sinus & ear involvement

• Two dairy calves die of respiratory disease. • The frontal sinus cavity contained pus (sinus infection). • BoCV was found in the sinus cavity, lymph nodes, salivary gland and inner ears. Case 3 – Sinus & vagus nerve involvement • Feedlot calf dies of bloat and a ruptured diaphragm. Necropsy also reveals some BRDC. • Frontal sinuses were congested and contained pink fluid loaded with BoCV. • BoCV was also detected in the VAGUS nerve • Tissues were also positive for BVDV. Case 4 – Eye involvement

• 10-day-old dairy calf dies of bovine coronavirus enterocolitis. • Although the eyes Diseased appeared normal, there Normal 3rd eyelid: Epithelial erosion, loss of goblet were loss of goblet cells, cells and lymphocytic infiltrates mild erosions and lymphocytic infiltrates in the 3rd eyelid. • BoCV was detected in the 3rd eyelid by IHC.

BoCV IHC of 3rd eyelid (above) Pink eye decision tree Question: Is it affecting a large number cattle in both eyes? Or a few animals in one eye

Many animals, Few animals, one quickly, in both eyes eye only? Has there be a significant Focus on sources of increase in flies and cattle trauma. Insects, tall ‘bunching?” grasses, head throwing YES NO from biting flies, kicking cows, tree and brush branches. Focus on contagious Focus on getting Possibly dust and pathogen (IBRV, cattle away from Coronavirus?) each other (usually a blowing bedding. fly, or heat problem). Spray flies. Case 5: Tonsil Feedlot animal dies of A majority of the tonsil is lined with attenuated epithelial cells. Mannheimia hemolytica The lumen contain sloughed BRDC. epithelial cells and hemorrhages

Cilia

Goblet

Portions of the tonsil are normal with mature ciliated No cilia, no goblet cells epithelial and goblet cells Case 5: Tonsil continued

Attached and sloughed epithelial cells stain positive for BoCV

BoCV IHC BoCV IHC Case 5: Dissolved mucus layer

400X, Mucicarmine stain, Sinus Healthy respiratory airways have a stable, consistent mucus layer (homogenous pink material)

400X, Mucicarmine stain, Tonsil The mucus layer is missing in this BoCV infected tonsil. Goblet cell One goblet cell remains Mucus and blood sloughs in the feces of cattle with enteric BoCV. Airway Normal Cilia Mature Goblet cells producing mucus

BoCV enterocolitis. Blood-stained mucoid feces. Necrotic crypts stain positive for BoCV by IHC BoCV in tissues from 18 cases of BRDC Nasal swabs and/or enteritis yield the most Nasal Inner ear Retrophar Mandib Salivary Sinus positive results Swab Lung Intestine swab Lymph N Lymph N Gland swab 1 23 32 (pool) 32.13 37.34 36.03 33.94 2 22 32 (pool) 34.81 36.11 35.66 28.24 3 25 24 neg 36.2 31.81 30.19 4 neg neg 20 (pool) neg 36 37 37 neg 5 22 36 20 (pool) 35 37 37 37 35 6 24 neg 20 (pool) 35 neg 39 neg 37 7 31 42(pool) 34 34 neg 36 8 38 42(pool) neg 38 neg neg 9 30 neg 35 neg 38 neg 10 27 34 38 neg neg neg 11 neg neg 34 34 neg neg 12 33 36 neg 39 neg neg 13 30 neg 34 34 37 neg neg 14 30 27 neg neg neg neg 35 15 28 18 (pool) neg 38.7 neg neg neg 16 33 18 (pool) neg neg neg neg neg 17 neg neg neg neg 33.7 37 36.5 neg 18 24.39 neg neg neg neg 35.4 32.1 29.8 Number 15/18 6/13 9/11 4/11 12/18 10/15 8/18 8/18 % positive 83% 46% 81% 36% 67% 67% 44% 44% Average Ct 28 35 25 34 36 37 36 33 BoCV in nerves Facial Olfactory Brain Trigeminal Optic Conjunct Vagus nerve nerve Ependyma Nerve nerve nerve 1 38.97 neg 2 neg neg 3 36.33 36.01 4 neg neg neg neg 5 40 neg neg 39 (susp) 6 neg neg neg neg 7 38 neg neg 8 neg neg neg 9 neg neg neg neg 10 neg neg neg neg 11 neg neg neg neg neg 12 neg neg neg neg 36 13 neg neg neg neg 14 34 neg neg 25 36 15 41 (susp) neg neg neg 16 neg neg neg neg 39 (susp) 17 neg neg neg neg 36.5 18 neg neg neg neg neg Number 6/18 1/18 0 1/11 1/12 3/4 1/4 % positive 33% 6% 0 9% 8% 75% 25% Average Ct 38 36 0 39 25 37 36 BoCV Diagnosis

Bovine Respiratory Sample consideration Comment Coronavirus Virus Features Shed Early: Peak sheding in a Sample at first sign of Avoid cattle that have been feedlot = 4 days post arrival disease treated multiple times Shed in high amounts Nasal swabs yield very Pooling up to 5 samples is high numbers of virus appropriate. The lab will do the pooling. Highly contageous If 20% of cattle are Virus levels after two weeks infected and you want are expected to be lower. to be 90% sure = sample 10 cattle Virus is stable when frozen Freeze swabs PCR finds virus, dead or alive. immediately. Can be If attempting to grow virus, frozen for months seek special media. Diagnostic Summary = If you nasal swab 10 cattle, early in the disease episode, and pool in 2 groups of 5, you can be about 90% sure of the diagnosis. Cost = $60 ($30 per pool of 5). The task of collecting and freezing nasal swabs can, and should, be done by the producer. Swabs

• Becton Dickenson Culturette swabs with liquid Stuarts media is a good, all-purpose swab. • Cotton swabs shipped in a red-topped tube with 1 ml of saline is also acceptable. • Avoid swabs with gel media as it can negatively impact a PCR reaction. • Avoid swabs with charcoal media. • If attempting to isolate (grow) a virus, then the VDL can send virus isolation media. Take-home messages 1. BoCV antibody titers, from either vaccination or natural exposure, don’t appear to last longer than 6 months. 2. BoCV infection appears to compromise the innate mucociliary apparatus of the upper respiratory tract. 3. IM (and likely SQ) vaccination with MLV prior to exposure appears to alleviate upper respiratory disease when given prior to exposure.