3. Treatment Group Allocation and Blinding
Litters were excluded from the study if the date of birth could not be established with reasonable accuracy (within ±3 days). Litters were followed up fortnightly until puppies were 6 weeks (42 days) old. If puppies changed ownership within the DSA during this period, the new owner was approached for enrollment of the puppy into the study. At 6 weeks of age, puppies, stratified by litter, were randomly allocated in a 1:1 ratio to receive a subcutaneous injection of either RV (1 mL of Defensor® 3, Zoetis, South Africa; batch numbers 139280, 139580, 164036, 152142, 170030, 221700, 230361) or placebo (1 mL of SW). Puppies were excluded if the allocation and intervention could not be done within ±3 days of 42 days of age. All puppies remaining in the study received a subcutaneous injection of RV at 13 weeks of age.
Prior to allocation, puppies were weighed and a blood sample collected (<5% of circulating blood volume, estimated as 85 mL/kg). Randomization was done by randomly allocating a treatment group (vaccine or placebo) to each of the series of radio-frequency identity microchips (each with a unique number) to be implanted in the puppies in the litter. One person in the study team, without knowledge of the treatment allocation, would select a microchip and implant it in a puppy. They would read the microchip number to a second person, who would refer to the allocation form of the microchip series for the treatment group code. The first person would select a preprepared syringe labeled with the treatment group code and inject it subcutaneously into the puppy. This process ensured allocation concealment and blinding of the owner to group allocation.
Owners were interviewed at baseline using a standardized questionnaire to determine type of puppy housing and reported puppy growth. Packed cell volume (PCV) and total protein (TP) were measured on the day of allocation (baseline) according to established protocols. RVNA titers were also measured at baseline (see below).
Puppies were followed up by weekly home visits or phone calls when owners were not at home. If puppies changed ownership within the DSA during this period, the new owner was approached for enrollment of the puppy into the study. Puppies who moved out of the DSA, or whose new owners did not consent to participate, were lost to follow up and censored on the date of exit. Owners were asked to report puppy deaths or disappearances immediately via phone call or text message. The study team visited these households to collect the carcass if available and conduct a verbal autopsy with an adult who was the primary caretaker of the dog [23]. Causes of death were classified as infectious, congenital/birth-related, trauma/accident, and unknown.
Blood samples for RVNA titers were collected at three time points: (i) 6 weeks of age (preinjection), (ii) 9 weeks of age (3 weeks after the first injection, and (iii) 16 weeks of age (3 weeks after the second injection. RVNA titers in serum samples were determined by the fluorescent antibody virus neutralization test (FAVN) performed at the Rabies Unit, Agricultural Research Council-Onderstepoort Veterinary Institute, according to the standard procedure described previously [5,24]. Subjects with a postvaccination RVNA titer level of at least 0.5 IU/mL were considered to have demonstrated an adequate response to vaccination, whereas those with a titer below 0.5 IU/mL did not show an adequate response. The titers of the dogs were established by comparison of the reactivity of the test sera to that of the OIE dog reference serum (at 0.5 IU/mL).
Q. If rabies shots last three years, why do puppies get one at four months and then again in a year?
A. Puppies and kittens are protected from disease for the first few weeks of their lives thanks to a “starter kit” of immunity passed from their mothers, primarily through their first feedings of an important kind of milk known as colostrum. The colostrum is nutrient-dense and provides newborns with not only antibodies to fight disease but also the beneficial bacteria essential to normal body functions.
Options for Vaccinating a Pregnant Dog for Rabies
One difference between rabies and other types of vaccines is that rabies vaccines are “killed” as opposed to live. As a result, it can be recommended by a veterinarian to be given during pregnancy, especially if its due to a safety. Your veterinarian may also advise you to give the vaccine if your state law on rabies vaccines mandates it and the vet has no leeway to allow you a legal delay. Even though a killed vaccine is safer, they still could result in a spontaneous abortion depending on the condition of the puppies in-utero and if it was given during the first four weeks of the pregnancy.
If your dog was previously vaccinated and is due again during her pregnancy, the best course of action is to wait until shes given birth to revaccinate her. Likewise if shes never been vaccinated for rabies prior to becoming pregnant, its safest to wait to administer the rabies vaccine if your vet agrees and you can legally do so.